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HEALTH  SCIENCES  STANDARD 


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QP51 4  .P45  An  intermediate  t 


Columbia  ^niberfiitpCo^vi^ 
College  of  pf)j»gicians  anb  ^urgeong 


i^eference  Itibrarp 


Presented  by 

k  DR.  WILLIAM  J.  OIESM 

*  fo  enrich  the  lihrary  resources 

av&ihbk  to  holders 
of  the 

GlES  FELLOWSHIP 

in  Biolosic&l  Chemistry 


PHYSIOLOGICAL  CHEMISTRY 


AN  INTERMEDIATE  TEXTBOOK 

OF 

PHYSIOLOGICAL  CHEMISTRY 

WITH   EXPERIMENTS 


BY 


C.  J.  V.  PETTIBONE,  Ph.D. 

ASSISTANT    PROFESSOR    OF    PHYSIOLOGICAL    CHEMISTRY,    MEDICAL    SCHOOL, 
UNIVERSITY  OF   MINNESOTA,   MINNEAPOLIS 


ST.  LOUIS 

C.  V.  MOSBY  COMPANY 

1917 


Copyright,    1917,    By    C.    V.    Mosby    Company 


Press  of 

C.   V.  Mosby  Company 

St.  Louis 


TO 
M.  A.  P.,  I.  P.  H.,  AND  M.  P.  G. 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 

Open  Knowledge  Commons  (for  the  Medical  Heritage  Library  project) 


http://www.archive.org/details/intermediatetextOOpett 


'    PREFACE 

My  aim  in  writing  this  book  has  been  to  prepare  an  inter- 
mediate text  which  would  cover  the  general  field  of  physiologi- 
cal chemistry  in  such  a  way  as  to  give  students  a  familiarity 
with  compounds  important  from  a  biochemical  viewpoint,  and 
to  acquaint  them  with  the  fundamental  processes  which  go  on 
in  the  animal  body.  I  have  attempted  to  avoid  confusing  the 
beginner  with  lengthy  discussions  of  debated  points,  but  to  set 
forth  as  clearly  as  possible  the  present  status  of  our  knowledge. 
The  material  is  so  chosen  that  the  book  may  be  used  for  inter- 
mediate classes,  or  for  advanced  work  if  supplemented  by  lec- 
tures. 

The  appended  laboratory  work  has  been  drawn  from  the 
manual  in  use  in  my  classes  for  the  last  five  years.  Much  of 
the  material  has,  of  course,  been  drawn  from  other  manuals. 

As  the  book  is  not  intended  to  be  an  advanced  text,  the 
micro  methods  for  the  analysis  of  urine  have  not  been. included. 

I  wish  to  acknowledge  suggestions  and  corrections  in  the 
laboratory  directions  from  various  colleagues,  particularly  Dr. 
F.  B.  Kingsbury.  I  wish  also  to  express  my  thanks  to  Dr.  W. 
H.  Hunter,  who  kindly  volunteered  to  read  the  manuscript  of 
the  theoretical  portion. 

C.  J.  V.  Pettibone. 

Miuneapolis,  Minn. 


CONTENTS 


PART  I 

CHAPTER  I 

Intkoductory 

Object  and  Importance  of  Physiological  Chemistry,  17;  Protoplasm,  18; 
Material  Bases,  19. 

CHAPTER  II 

Elements,  Inorganic  Materials,  Water 

Elements  Found  in  the  Body,  21 ;  Importance  Not  Determined  by 
Amount  Present,  21;  Carbon,  Hydrogen,  Oxygen,  Nitrogen,  Sulphur,  and 
Phosphorus,  22;  Sodium,  Potassium,  Calcium,  Magnesium,  and  Iron,  24; 
Chlorine,  Iodine,  Fluorine,  Etc.,  26;  water,  26. 

CHAPTER  III 

Carbohydrates 

Composition,  Occurrence,  General  Function,  27;  Structure  of  the  Carbo- 
hydrates, 27;  Optical  Activity,  28;  Classification  of  Carbohydrates,  34; 
Origin  and  Synthesis,  35 ;  Interconversion  of  Carbohydrates,  37 ;  Combina- 
tion of  Carbohydrates  with  One  Another,  and  with  Other  Substances,  38; 
Behavior  with  Strong  Alkalies,  38;  Behavior  with  Acids,  39;  Oxidation 
of  Carbohydrates,  40;  Reduction  of  Carbohydrates,  42;  Formation  of  Osa- 
zones,  43 ;  The  Molisch  Test,  44 ;  Fermentation — Enzymes,  44 ;  Nomenclature 
and  Classification,  46;  Specific  Nature,  47;  Influence  of  Temperature,  47; 
Effect  of  Chemical  Reaction,  47;  Reversibility,  48;  Active  and  Inactive 
Form,  48 ;  Action  Retarded  by  Products,  48 ;  Progressive  Action,  48 ;  Sum- 
mary, 49 ;  Individual  Groups  of  Carbohydrates,  49 ;  Pentoses,  49 ;  Absorp- 
tion Spectra,  50;  Hexoses.  CgHjjOg,  51;  Glucose,  51;  Fructose.  (Levulose, 
Fruit  Sugar),  52;  d-Galactose,  52;  Amino  Sugars,  53;  d-Glucuronic  Acid, 
53;  Disaccharides,  54;  Saccharose,  (Sucrose,  Cane  Sugar),  54;  Lactose,  56; 
Maltose,  57;  Polysaccharides,  57;  Starch  (GJI,„0,)^.,  58;  Dextrins,  59; 
Inulin,  59;  Gums  and  Mucilages,  59;  Cellulose,  59;  Glycogen,  60;  Gluco- 
sides,  61. 

11 


12  CONTENTS 

CHAPTER  IV 

Fats,  Phosphatids,  and  Allied  Substances 

Distribution  and  Importance,  62 ;  Composition  and  Structure,  62 ;  Gen- 
eral Properties,  64 ;  Emulsification,  64 ;  Saponification,  65 ;  Rancid  Fats,  66 ; 
Detection  and  Identification,  66;  Important  Fats,  68;  Lecithin  and  Choles- 
terol, 69;   Lecithin,  69;   Cholesterol,  70. 

CHAPTER  V 

Proteins 

Introductory,  72  ;  Elementary  Composition,  72  ;  Classification,  73  ;  Prep- 
aration of  Proteins  from  Materials  in  Which  Tliey  Occur,  74;  Molecular 
Weight,  75;  Hydrolysis,  75;  Amino  Acids  Obtained  by  Hydrolyzing  Pro- 
tein, 76;  General  Properties  and  Reactions  of  Amino  Acids,  79;  General 
Protein  Reactions,  82;  Color  Tests,  82;  Precipitation  Reactions,  84;  Col- 
loids, 84;  Classification  and  Properties  of  Colloids,  84;  Tyndall's  Phe- 
nomenon, 86 ;  Electrical  Properties  of  Colloids,  86 ;  Methods  of  Precipitating 
Colloids,  86;  Structure  of  the  Protein  Molecule,  89;  Putrefaction  of  Pro- 
teins, 93 ;  Individual  Groups,  Simple  Proteins,  95 ;  Albumins^  95 ;  Globulins, 
95;  Glutelins,  96;  Prolamines,  96;  Albuminoids,  96;  Histones,  97;  Pro- 
tamines, 97;  Conjugated  Proteins,  98;  Glycoproteins,  98;  Phosphoproteins, 
98;  Hemoglobins,  100;  Detection  of  Hemoglobin,  102;  Absorption  Spectra 
of  Oxyhemoglobin  and  Hemoglobin,  104 ;  Derivati-ves  of  the  Hemoglobins, 
105;  Fate  of  Blood  Pigment  in  the  Body,  107;  Nucleoproteins,  107;  Lecitho- 
proteins,  109 ;  Derived  Proteins,  109 ;  Primary  Protein  Derivatives,  109 ; 
Secondary  Protein  Derivatives,  111. 

CHAPTER  VI 

Some  Familiar  Foodstuffs — Some  Important  Tissues 

Some  Important  Foodstuffs,  114;  Cooking  and  Preparation  of  Foods, 
115;  Milk,  115;  Butter,  116;  Cheese,  117;  Meats,  117;  Eggs,  117;  Vege- 
tables, 117;  Breadstuffs,  118;  Choice  of  Diet,  118;  Some  Important"  Tis- 
sues, 119 ;  Muscle,  119 ;  Brain  and  Nerves,  120 ;  Bones  and  Teeth,  121 ;  Con- 
nective Tissue,  121 ;  The  Blood,  121 ;  Reaction  of  the  Blood,  123 ;  Osmotic 
Pressure,  124;  Coagulation  of  the  Blood,  124;  Lymph,  125;  Tlie  Skin,  125. 

CHAPTER  VII 

Digestion  in  the  Mouth 

General  Purpose  of  Digestion,  126;  Preparation  of  Food,  127;  Saliva, 
127. 


CONTENTS  _  13 

CHAPTER  VIII 

Digestion  in  the  Stomach 

Importance,  131;  Methods  of  Study,  131;  Causation  of  Flow  of  Gastric 
Juice,  132;  General  Character  of  the  Secretion,  134;  Hydrochloric  Acid, 
134;  Source  of  the  Hydrochloric  Acid,  136;  The  Functions  of  the  Hydro- 
chloric Acid,  136;  Enzynfes  of  the  Gastric  Juice,  137;  Products  of  Peptic 
Digestion,  138;  Eennin,  138;  Are  Pepsin  and  Rennin  Identical?,  139;  The 
Stomach  Wall  is  Not  Digested,  140;  Passage  of  the  Food  Into  the  Intes- 
tine, 140. 


CHAPTER  IX 

Digestion  in  the  Intestine 

General,  141 ;  Pancreatic  Juice,  141 ;  Mechanism  6i  Flow,  142 ;  Com- 
X^osition  of  Pancreatic  Juice,  142;  Trypsin,  143;  Eennin,  144;  Action  on 
Fats,  144;  Action  on  Starches,  144;  The  Bile,  145;  Causes  of  Flow,  145; 
Composition,  146 ;  Bile  Pigments,  146 ;  Bile  Salts,  147 ;  Intestinal  Secretion, 
147;  Erepsin,  148;  Other  Enzymes,  148;  Excretory  Function  of  Intestinal 
Secretion,  148;   Bacterial  Action  in  the  Intestine,  149;  Feces,  150. 


CHAPTER  X 

Absorption 

General,  152 ;   Absorption  of  Proteins,  153 ;   Carbohydrate  Absorption, 
,  153 ;  Absorption  of  Fats,  154. 


CHAPTER  XI 

Urine 

General,  155;  Physical  Properties,  156;  Volume,  156;  Color,  Trans- 
parency, 157;  Consistency,  Odor,  Taste,  158;  Specific  Gravity,  158;  Total 
Solids,  158;  Optical  Activity,  Reducing  Power,  Fermentation,  Etc.,  159; 
Reaction,  160;  Urea,  161;  Uric  Acid  and  Other  Purine  Derivatives,  163; 
Hippuric  Acid,  166;  Ammonia,  167;  Creatinine  and  Creatine,  167;  In- 
organic Constituents,  169;  Chlorides,  169;  Phosphates,  170;  Sulphates,  170; 
Carbonates,  171;  Sodium,  Potassium,  Calcium,  and  Magnesium,  171;  Patho- 
logical Constituents  of  the  Urine,  172. 


14  CONTENTS 

CHAPTER  XII 

Metabolism 

General,  173;  Protein  Metabolism,  174;  Amount  of  Protein  Eequired, 
176;  Nitrogen  Balance,  176;  Carbohydrate  Metabolism,  182;  Sources  of 
Glycogen,  187;  Metabolism  of  Fats,  189;  Metabolism  of  Inorganic  Material, 
190 ;  Energy  Exchange,  191 ;  Utilization  of  Alcohol  by  the  Body,  195 ;  Star- 
vation, 196;  Unknown  Food  Constituents,  197;  Vitamines,  197;  Body  Tem- 
perature, 199;  Influence  of  Organs  of  Internal  Secretion,  199. 

PART  II 
LABORATOEY  WORK 

Materials  for  laboratory  work,  202. 

CHAPTER  I 

General  Laboratory  Instructions 
General  Laboratory  Instructions,  208. 

CHAPTER  II 

Detection  of  the  Elements  and  of  Inorganic  Salts 

Carbon,  210;  Hydrogen,  210;  Oxygen,  210;  Nitrogen,  211;  Sulphur, 
211;  Preparation  of  Muscle  Extract,  211;  Muscle  Residue,  212;  Blood 
Serum,  212;  Bone,  213;  Tests  for  Inorganic  Materials,  213;  Chlorides,  213; 
Sulphates,  214;  Phosphates,  214;  Carbonates,  214;  Calcium,  214;  Mag- 
nesium, 214;  Iron,  215;  Sodium,  215;  Potassium,  216. 

CHAPTER  III 

Carbohydrates 

Monosaccharides,  217;  Dextrose,  217;  Levulose,  221;  Galactose,  221; 
Arabinose,  222;  Disaccharides,  222;  Saccharose  (Sucrose),  222;  Maltose, 
223;  Lactose,  224;  Polysaccharides,  224;  Starches,  224;  Dextrines,  225;  Gly- 
cogen, 226. 

CHAPTER  IV 

Fats  and  Phosphatids 
Fats,  227;  Phosphatids,  Lecithin,  230. 


CONTENTS  15 

CHAPTER  V 

Proteins 

General  Protein  Reactions,  232 ;  Color  Reactions,  232 ;  Precipitation 
Tests,  234;  Individual  Groups — Simple  Proteins,  237;  Albumins,  237;  Globu- 
lins, 238;  Prolamines,  240;  Glutelius,  240;  Albuminoids,  240;  Histones,  241; 
Protamines,  242;  Conjugated  Proteins,  242;  Glycoproteins,  242;  Hemo- 
globins, 243;  Phosphoproteins,  250;  Nucleoproteins,  251;  Lecithoproteins, 
253;  Derived  Proteins,  253;  Primary  Protein  Derivatives,  Proteans,  253; 
Metaproteins,  253 ;  Secondary  Protein  Derivatives,  255 ;  Proteoses,  255  ;  Pep- 
tones, 256;  Peptids,  257;  Amino  Acids,  257. 

CHAPTER  VII 
Salivary  Digestion 
Conij)osition,  260;  Digestive  Action,  261. 

CHAPTER  VIII 

Gastric  Digestion 

Preparation  of  Artificial  Gastric  Juice,  264;  Composition  of  Gastric 
Juice,  264;  Digestive  Action  of  Gastric  Juice,  266;  Motor  Power  of  the 
Stomach,  268;  Rate  of  Absorption  From  the  Stomach,  268. 

CHAPTER  IX 

Pancreatic  Digestion — Bile 

Pancreatic  Juice,  269;  Composition  of  Pancreatic  Juice,  269;  Diges- 
tive Action,  269;  Intestinal  Juice,  270;  Bile,  270;  Composition,  270;  Effect 
of  Bile  on  Surface  Tension,  271;  Biliary  Calculi,  or  Gall  Stones,  271. 

CHAPTER  XI 

Qualitative  Study 

Urinb 

Inorganic  Constituents,  273;  Organic  Constituents,  275;  Collection  and 
Preservation  of  a  Specimen  for  Quantitative  Analysis — General  Properties, 
278;  Quantitative  Analysis,  281;  Total  Solids,  281;  Acidity,  281;  Total 
Nitrogen  (Kjeldahl  Method),  287;  Ammonia,  290;  Urea,  292;  Uric  Acid, 
293;  Hippuric  Acid,  294;  Purine  Bases,  295;  Creatinine  (Folin),  295; 
Indican,  296;  AUantoin,  297;  Oxalic  Acid,  297;  Chlorides,  297;  Sulphates, 
298;  Phosphates,  299;  Pathologic  Urine,  299;  Proteins,  299;  Carbohy- 
drates, 303;  Acetone  Bodies,  306. 


16  CONTENTS 

APPENDIX 

Directions  for  Making  Up  Quantitative  ok  Speciai;  Eeagents 

Ammonium  Thiocyanate,  Standard,  for  Chlorides,  308;  Barfoed's  Solu- 
tion, 308;  Barium  Chloride  for  Sulphate  Determination,  308;  Benedict's 
Solution  for  Carbohydrate  Estimation,  309;  Congo  Paper,  309;  Esbach's 
Reagent,  309;  Fehling's  Solution  (Quantitative),  309;  Fehling's  Solution 
(Qualitative),  309;  Folin-Schaffer  Eeagent,  309;  Formalin  (Neutral),  309; 
Glyoxylic  Acid  Solution,  310;  Guenzburg's  Reagent,  310;  Magnesia  Mix- 
ture, 310;  Mett's  Tubes,  310;  Millon's  Reagent,  310;  Molisch's  Reagent, 
311;  Nylander's  Reagent,  311;  Pancreatic  Solution  (''Artificial  Pancreatic 
Juice"),  311;  Pepsin  Solution  (Artificial  Gastric  Juice),  311;  Potassium 
Bichromate  N/2,  311;  Potassium  Permanganate  N/20,  311;  Silver  Nitrate 
(Standard)  for  Volhard  Chloride  Method,  311;  Special  Sodium  Acetate  Solu- 
tion (for  Uranium  Acetate  Method  for  Phosphates),  311;  Stokes'  Reagent, 
311;   Toepfer's  Reagent,  312;  Uranium  Acetate  Solution   (Standard),  312. 


PHYSIOLOGICAL  CHEMISTRY 


PART  I 


CHAPTER  I 

INTRODUCTORY 

The  scientific  field  known  variously  as  Physiological,  Biologi- 
cal or  Biochemistry  is  the  branch  of  science  which  treats  of  the 
chemical  constitution,  reactions  and  products  of  living  ma- 
terial, whether  of  animal  or  plant  origin.  There  is  a  growing 
tendency  to  use  the  terms  Biological  and  Biochemistry  to  de- 
note the  entire  field,  and  to  restrict  the  term  Physiological 
chemistry  to  that  portion  of  the  subject  dealing  with  animal 
material,  but  this  practice  is  by  no  means  general.  It  was  once 
believed  that  the  chemical  processes  going  on  in  plants  and  ani- 
mals were  fundamentally  different.  Synthesis  or  building  up 
was  considered  characteristic  of  plants,  whereas  animals  were 
known  to  desynthesize  or  break  down  the  substances  which  they 
ate.  We  now  know  that  this  difference  is  a  quantitative  and 
not  a  qualitative  one,  for  if  kept  in  the  dark,  plants  take  up 
oxygen,  burn  their  constituents  and  give  off  carbon  dioxide  in 
a  manner  analogous  to  the  process  predominating  in  animals. 
Animals,  on  the  other  hand,  are  now  known  to  be  capable  of 
performing  numerous  and  elaborate  syntheses,  breaking  down 
the  materials  of  their  food  to  simpler  compounds,  but  rebuild- 
ing many  of  the  fragments  into  tissue  substance,  or  altering 
them  to  produce  compounds  having  specialized  biological  func- 
tions. 

17 


18  PHYSIOLOGICAL    CHEMISTRY 

Object  and  Importance  of  Physiolog-ical   Chemistry. — The 

ultimate  object  of  workers  in  the  field  is  to  establish  a  rela- 
tionship between  chemical  composition  and  biological  function, 
to  be  able  to  explain  the  workings  of  cells  or  of  the  various  or- 
gans and  tissues  in  terms  of  chemical  reactions,  but  experi- 
mentors  are  still  far  from  the  attainment  of  this  end,  although 
many  problems  now  are  clearly  understood  which  a  few  year's 
ago  were  still  unsolved.  The  findings  of  physiological  chem- 
ists, and  the  methods  of  analysis  developed  by  them  have  been 
of  the  greatest  value  to  the  science  of  medicine  in  general,  and 
to  the  medical  practitioner  in  particular.  Since  the  body  is 
made  up  of  chemical  compounds,  and  since  many  of  its  activ- 
ities depend  upon  chemical  reactions,  it  is  obvious  that  any 
light  thrown  upon  the  nature  and  properties  of  its  components 
will  tend  to  make  clearer  the  character  of  the  reactions  in- 
volved in  the  normal  functioning  of  the  tissues,  thus  furnishing 
a  basis  for  the  study  and  correction  of  abnormal  or  diseased 
conditions.  Both  diagnosis  and  treatment  have  come  to  depend 
more  and,  more  upon  the  findings  of  the  physiological  chem- 
ists, and  the  general  advancement  of  medicine  has  been  greatly 
furthered  by  the  results  of  biochemical  research. 

Protoplasm. — Living  material,  whether  of  plant  or  animal 
origin  has  been  found  to  consist  of  a  substance  which  is  strik- 
ingly uniform  throughout  the  entire  living  world.  This  ma- 
terial has  been  given  the  name  protoplasm  (from  the  Greek 
words  meaning  "first,"  and  ''form").  It  is  a  jelly-like 
watery  mass,  sometimes  fairly  rigid  in  form,  possessing  cer- 
tain characteristics  which  serve  to  distinguish  living  from  life- 
less material.  The  first  of  these  is  the  power  of  growth, — 
growth  from  internal  forces  such  as  we  observe  in  animals  and 
plants,  and  not  growth  from  without  such  as  the  enlarging  of 
a  crystal.  The  second  is  the  power  of  respiration,- — taking  up 
oxygen  and  giving  off  carbon  dioxide.  The  third  is  the  power 
of  movement, — from  place  to  place  in  animals,  and  movement 
incident  to  growth  in  plants.  The  fourth  is  irritability,  and  the 
fifth  the  power  of  reproduction.     All  living  material  possesses 


INTRODUCTION  19 

these  five  properties,  and  no  lifeless  material  possesses  all  of 
them.  Physiological  chemistry  may  be  looked  upon  as  the 
study  of  the  chemistry  of  protoplasm,  its  products  and  the  sub- 
stances which  it  requires  for  the  continuance  of  its  normal 
functions. 

Material  Bases. — Amounts  in  Body. — In  beginning  the  study 
of  so  broad  and  complicated  a  field  it  is  dii^cult  to  choose  a 
point  of  attack.  The  most  satisfactory  plan  will  be  first  to  be- 
come familiar  with  the  chemical  substances  out  of  which  living 
material  is  made  up.  The  number  of  these  compounds  is  nat- 
urally large,  but  for  convenience  they  may  be  classified  in  five 
great  groups  which  are  given  the  name  of  the  Base  Materials, 
or  Material  Bases. 

The  five  groups  are  as  follows : 

I.  Inorganic  materials  including  water. 

II.  Carbohydrates. 

III.  Fats,  Phosphatids  and  related  compounds. 

IV.  Proteins. 

v.  Extractives. 

Our  first  task  will  be  to  become  familiar  with  the  character- 
istics and  properties  of  the  Material  Bases,  studying  group 
reactions  and  also  the  specific  properties  of  important  individ- 
ual compounds,  and  methods  for  their  detection  and  estima- 
tion. We  will  then  trace  the  history  of  the  various  substances 
in  their  passage  through  the  body,  considering  their  fate  in 
the  alimentary  canal,  their  subsequent  behavior  as  constitu- 
ents in  the  body  tissues  and  fiuids,  and  the  final  elimination  of 
end  products  formed  by  their  destruction. 

The  relative  amounts  of  the  different  classes  of  Material 
Bases  in  the  animal  body  are  somewhat  variable.  Water  and 
other  inorganic  materials  make  up  about  65-70%  of  the  entire 
body  weight  of  which  only  4.5-5%  is  ash  and  the  remainder 
water.  Carbohydrates  are  present  only  in  small  quantities,  the 
amount  being  less  than  1%.  Fats  and  related  compounds  vary 
considerably  in   amount  with  the   general   state   of  the  body. 


20  PHYSIOLOGICAL    CHEMISTRY 

since  they  may  be  stored  away  in  large  quantities.  The  body 
contains  on  the  average  about  15%.  Proteins  vary  less  from 
an  absolute  standpoint,  but  relatively  the  percentage  depends 
upon  the  amount  of  fat.  The  amount  in  the  body  averages 
also  about  15%.  Extractives,  a  heterogeneous  group  of  com- 
pounds classed  together  because  they  are  water  soluble,  and 
belong  in  none  of  the  other  classes,  make  up  less  than  1% 
of  the  body  weight.  This  group  will  receive  no  further  treat- 
ment as  such,  but  the  substances  included  in  it  (urea,  creat- 
inine, etc.)  will  be  discussed  individually  in  connection  with 
the  tissues  or  fluids  in  which  they  occur. 


CHAPTER   II 
ELEMENTS,    INORGANIC   MATERIALS,   WATER 

Elements  Found  in  the  Body. — The  body  is  made  up  of  a 
large  number  of  chemical  elements  which  are  present  in  very 
unequal  amounts,  and  distributed  quite  unevenly  in  the  vari- 
ous tissues  and  body  fluids.  Certain  of  these  elements  are  in 
all  living  cells,  others  only  in  particular  kinds  of  cells  or  in 
particular  animals.  Still  others  are  present  only  accidentally 
or  temporarily.  The  elements  found  most  frequently  are  the 
non-metals  carbon,  hydrogen,  oxygen,  nitrogen,  sulphur,  and 
phosphorus ;  the  metals  sodium,  potassium,  calcium,  magnesium 
and  iron;  the  halogens,  chlorine,  iodine,  and  fluorine.  In  addi- 
tion, there  are  many  other  elements  such  as  silicon,  copper, 
manganese,  arsenic,  silver,  lead,  bromine,  lithium,  etc.,  found 
only  in  traces  or  only  in  a  few  animals. 

Importance  Not  Determined  by  Amount  Present. — Three  of 
the  elements,  carbon,  hydrogen  and  oxygen  alone  make  up 
over  90%  of  the  body  weight.  The  conclusion  should  not  be 
drawn,  however,  that  these  are  the  only  important  elements  and 
that  those  elements  or  compounds  present  in  small  amounts  or 
traces  are  relatively  unimportant  to  the  organism.  Quite  the 
contrary  may  be  the  case.  The  body  of  an  average-sized  adult 
man  contains  only  about  three  grams  of  iron,  and  yet  this  is  so 
necessary  to  life  that  an  animal  fed  for  some  time  on  a  diet 
which  contains  no  iron  will  die  quite  as  surely,  though  of 
course  not  as  quickly,  as  if  it  had  been  deprived  of  food  alto- 
gether. The  principle  involved  may  be  formulated  as  the  Law 
of  the  Minimum  which  states  that  the  importance  of  a  given 
substance  to  the  animal  organism  is  independent  of  the  rela- 
tive amount  in  which  it  is  required.  Striking  examples  of  this 
principle  have  developed  in  recent  years,   and  we  now  know 

21 


22  PHYSIOLOGICAL    CHEMISTRY 

that  the  body  requires  certain  compounds  of  which  nothing 
was  known  a  few  years  ago.  Some  of  these  substances  appear 
to  be  organic  compounds.  Although  the  amounts  of  these  ma- 
terials of  unknown  constitution  which  are  required  by  the 
body  are  only  a  small  fraction  of  a  gram,  still  their  absence 
from  the  diet  will  cause  severe  disorders  and  ultimate  death. 

The  importance  to  the  body  of  the  individual  elements  ex- 
tends much  beyond  the  function  of  serving  as  inert  building 
stones  out  of  which  the  body  materials  are  made  up.  Many  of 
the  chemical  reactions  in  the  body  are  greatly  influenced  or 
modified  by  certain  of  the  elements.  For  example,  two  important 
processes,  the  clotting  of  the  blood  which  tends  to  protect  a 
wounded  animal  from  bleeding  to  death,  and  the  clotting  of  milk 
in  the  stomach,  the  first  step  in  its  digestion,  are  dependent  upon 
the  presence,  among  other  things,  of  calcium,  and  without  this 
metal  neither  of  these  reactions  can  occur. 

The  accompanying  table  gives  a  survey  in  round  numbers  of 
the  relative  amounts  of  the  elements  present  in  the  body. 

Carbon    17.5 

Hydrogen    10.2 

Oxygen 66 

Nitrogen    2.4 

Sulphur 0.2 

Phosphorus   0.9 

Sodium    0.3 

Potassium 0.4 

Calcium  1.6 

Magnesium    0.05 

Iron    0.005 

Chlorine 0.3 

Iodine    Traces 

Fluorine    Traces 

Other  elements    Traces 

Carbon,  Hydrogen,  Oxygen,  Nitrogen,  Sulphur  and  Phos- 
phorus.— Carbon  has  the  property  of  forming  a  very  large  num- 


ELEMENTS,    INORGANIC    MATERIALS,    WATER  23 

ber  of  compounds  with  hydrogen,  oxygen,  and  sometimes  sul- 
phur, phosphorus  and  other  elements.  These  compounds  arc 
so  numerous  that  a  separate  branch  of  chemistry  is  devoted  to 
them  under  the  name  of  organic  chemistry.  Thirty  per  cent  or 
more  of  the  body  consists  of  organic  material,  or  compounds 
of  carbon  with  hydrogen,  oxygen,  etc.  The  importance  of  these 
compounds  is  so  great  that  special  chapters  will  be  devoted  to 
the  important  groups.  The  body  receives  its  carbon  compounds 
in  the  foods,  the  solid  portion  of  which  is  very  largely  organic 
material.  Aside  from  the  organic  components  of  the  tissues, 
carbon  is  also  found  in  inorganic  form,  chiefly  as  calcium  car- 
bonate in  bone,  and  as  sodium  bicarbonate  in  the  blood.  This 
latter  compound  is  of  great  importance,  since,  although  its 
solution  is  neutral,  it  has  the  power  of  neutralizing  acids,  thus 
protecting  the  tissues  when  acids  are  introduced  into  the  blood 
either  from  the  digestive  tract  or  as  the  result  of  the  breaking 
down  of  body  materials.  Carbon  is  eliminated  from  the  body 
as  carbon  dioxide,  given  off  through  the  lungs,  or  as  more  com- 
plex compounds  such  as  urea  and  other  materials  excreted  in 
the  urine  or  the  feces.  If  heated,  organic  compounds  char, 
leaving  a  black  residue  of  carbon. 

Hydrogen  and  oxygen  aside  from  being  constituents  of  water, 
organic  and  inorganic  compounds,  also  play  more  specialized 
roles  in  the  body.  Oxygen,  which  makes  up  about  two-thirds 
of  the  entire  body  w^eight  is  taken  up  as  a  constituent  of  the 
compounds  in  the  food  and  in  the  process  of  respiration.  Hy- 
drogen, the  presence  of  which  in  ionic  form  is  the  distinguishing 
characteristic  of  acids,  plays  an  important  part  in  the  digestion 
of  proteins  by  gastric  juice.  Hydrogen  and  oxygen  are  elimi- 
nated from  the  body  chiefly  as  water  and  CO2  given  off  through 
the  lungs,  and  as  w^ater  and  in  organic  compounds  in  the  urine 
and  feces.  On  heating  organic  materials  they  decompose,  and 
the  hydrogen  and  oxygen  are  given  off  as  water  and  various 
other  compounds. 

Nitrogen,  sulphur  and  phosphorus  also  play  various  roles  in 
the  body  and  all  three  are  found  both  in  organic  and  inorganic 


24  PHYSIOLOGICAL    CHEMISTRY 

compounds.  Nitrogen  is  a  constituent  of  all  proteins.  Many 
proteins  contain  sulphur  and  phosphorus  as  well.  Nitrogen 
occurs  in  various  other  compounds  throughout  the  body,  and 
the  body  fluids  contain  gaseous  nitrogen  in  solution,  as  is  to  be 
expected  since  nitrogen  is  somewhat  soluble  in  an  aqueous 
liquid,  and  in  the  lungs  the  blood  comes  in  contact  with  the  air 
which  is  made  up  about  four-fifths  of  nitrogen.  So  far  as  is 
known  this  dissolved  nitrogen  has  no  influence  on  the  body's 
activities.  Nitrogen  is  excreted  mainly  as  urea,  uric  acid  and 
other  products  formed  by  the  decomposition  of  proteins  in 
the  body.  If  heated  with  soda  lime,  an  organic  substance  of 
this  type  gives  off  its  nitrogen  as  ammonia.  Sulphur  and  phos- 
phorus are  present  in  various  compounds  other  than  the  pro- 
teins. Of  these  the  phosphatids  are  perhaps  most  interesting. 
This  group  will  be  considered  later.  Calcium  phosphate  makes 
up  about  85%  of  the  ash  of  bone.  Sodium  phosphate  is  found 
in  the  blood  and  tissues.  Sulphur  and  phosphorus  are  excreted 
mainly  in  the  urine  as  sulphates  and  phosphates,  but  also  in  the 
feces.  Unoxidized  sulphur  may  be  split  off  from  organic  com- 
pqjinds  by  boiling  with  alkali.  On  adding  lead  acetate,  a  dark 
precipitate  of  lead  sulphide  will  form.  This  test  may  be  con- 
firmed by  adding  an  acid.  Hydrogen  sulphide  will  be  given 
off  and  may  be  identified  by  a  paper  moistened  with  lead  acetate 
on  which  lead  sulphide  will  form  as  a  shiny  dark  precipitate. 
Sulphates  are  detected  by  precipitating  as  barium  sulphate. 
Phosphates  are  detected  by  precipitating  as  ammonium  phospho- 
molybdate. 

Sodium,  Potassium,  Calcium,  Magnesium  and  Iron. — These 
metals  make  up  only  a  small  part  of  the  body,  but  they  are  none 
the  less  important.  They  are  distributed  widely  throughout  the 
body  tissues  and  fluids.  There  usually  is  more  sodium  than 
potassium  in  body  fluids,  and  more  potassium  than  sodium  in 
the  solid  tissues.  These  metals  are  present  as  chlorides,  sul- 
phates or  phosphates.  Sodium  chloride  in  the  blood  is  interest- 
ing since  it  furnishes  chlorine  for  the  hydrochloric  acid  of  the 
gastric  juice,  whereas  sodium  bicarbonate  is  very  important  in 


ELEMENTS,    INORGANIC    MATERIALS,    WATER  25 

preserving  the  neutrality  of  the  blood  as  above  noted.  Sodium 
chloride  is  the  only  inorganic  substance  which  is  contained  in 
a  mixed  diet  in  amounts  insufficient  for  the  body's  needs.  Ac- 
cordingly our  food  must  be  salted.  Herbivorous  animals,  living 
on  plants  in  which  potassium  is  in  excess  of  sodium,  crave  salt, 
and  it  is  well  known  that  cattle  must  be  "salted"  to  keep  them 
in  good  health.  Sodium  and  potassium  also  affect  the  irritabil- 
ity of  muscle  and  nerve  tissue  so  that  their  role  in  the  organism 
may  perhaps  include  little  understood  regulatory  functions. 
These  metals  may  be  detected  by  the  flame  test  or  by  precipita- 
tion as  sodium  pyroantimonate  or  potassium  cobaltinitrite. 

Calcium  and  magnesium,  while  found  in  all  cells  in  the  body, 
are  present  in  largest  amount  in  the  bones  and  teeth,  which 
contain  about  99%  of  all  the  calcium,  and  70%  of  all  the  mag- 
nesium in  the  body.  Their  phosphates  and  carbonates  make  up 
about  98.5%  of  all  the  inorganic  material  in  bone.  That  these 
metals  have  other  roles  to  play  in  the  body,  however,  is  demon- 
strated by  the  failure  of  blood  and  milk  to  clot  in  the  absence 
of  calcium.  Calcium  is  detected  by  precipitation  as  calcium 
oxalate.  Magnesium  is  present  in  much  smaller  amounts  than 
calcium,  and  less  is  known  of  its  physiological  activities.  It  is 
interesting  in  this  connection  that  magnesium  sulphate  acts  as 
an  ansesthetic  on  mammals  and  that  it  paralyzes  the  endings  of 
the  motor  nerves  in  the  muscles.  Magnesium  is  detected  by 
precitation  as  magnesium  ammonium  phosphate. 

Iron,  though  present  only  to  the  amount  of  a  few  grams  in 
the  body  of  an  adult  man,  still  is  distributed  very  widely.  Its 
most  conspicuous  role  is  in  connection  with  the  hemoglobin  of 
the  blood,  of  which  it  is  a  constituent.  This  substance  carries 
oxygen  from  the  lungs  to  the  tissues.  Iron  probably  is  pres- 
ent also  in  traces  of  inorganic  iron  compounds.  The  spleen 
contains  relatively  much,  so  that  it  has  been  suggested  that  the 
spleen  has  some  role  to  play  in  connection  with  the  iron  com- 
pounds of  the  blood.  Also  the  liver  is  concerned  with  the  fate 
of  hemoglobin,  and  serves  as  a  clearing  house  through  which 
hemoglobin  from  worn  out  corpuscles  is  broken  down  and  ex- 


26  PHYSIOLOGICAL    CHEMISTRY 

creted  by  way  of  the  bile  into  the  intestine.  Iron  is  also  ex- 
creted in  the  urine,  the  amount  being  no  greater  than  10  or  11 
mg.  per  liter. 

Iron  is  detected  by  precipitation  as  ''Prussian  Blue"  or  fer- 
ric ferrocyanide  or  by  the  formation  of  ferric  thiocyanate  which 
is  red  in  color. 

Chlorine  Iodine,  Fluorine,  etc. — Chlorides  are  present  in 
traces  in  all  the  tissues  and  fluids ;  the  blood  contains  about  0.4% 
sodium  chloride,  the  gastric  juice  about  0.4%  hydrochloric  acid. 
The  necessity  of  adding  sodium  chloride  to  the  diet  has  already 
been  referred  to.  Chlorides  are  excreted  in  the  urine  in 
amounts  varying  with  the  amount  in  the  food,  10-12  gms. 
daily  being  an  average  figure.  Chlorine  may  be  detected  by 
precipitation  as  silver  chloride.  Iodine  is  interesting  chiefly  in 
connection  with  its  occurrence  in  the  thyroid  gland.  This  gland, 
by  means  of  a  compound  which  it  produces  and  pours  out  into 
the  blood  stream,  has  far  reaching  influence  on  the  chemical 
reactions  going  on  in  the  body.  This  important  substance  is  an 
iodine  compound.  If  an  iodide  gets  into  the  blood  stream,  the 
salivary  glands  have  the  power  of  excreting  it.  After  taking  a 
capsule  containing  potassium  iodide,  iodine  may  be  demonstrated 
in  the  saliva.     Fluorine  is  found  in  the  bones  and  teeth. 

Other  elements  may  occur  either  regularly  or  accidentally 
in  the  body  tissues  or  fluids,  but  usually  only  in  traces. 

Water. — Water  makes  up  about  %  of  the  body  weight  of 
mammals,  and  a  much  larger  part  in  some  lower  animals.  All 
tissues  contain  it,  even  the  enamel  of  the  teeth:  blood,  lymph, 
the  digestive  juices,  urine,  etc.,  are  %o-%o  water;  the  organs 
and  softer  tissues  about  %.  Water  serves  a  variety  of  func- 
tions. It  is  the  circulating  medium  for  transporting  food  and 
Avaste  material  to  and  from  the  cells,  it  holds  various  sub- 
stances in  solution  and  makes  ionization  possible,  and  is  a 
medium  for  the  excretion  of  w^aste.  It  also  distributes  the  heat 
of  the  body,  and  by  evaporation  from  the  skin  is  instrumental 
in  regulating  body  temperature.  An  animal  may  survive  for 
many  days  without  nourishment,  but  if  water  also  is  withheld, 
death  follows  in  a  few  days'  time. 


CHAPTER  III 
CARBOHYDRATES 

Composition,  Occurrence,  General  Function. — The  carboliy- 
drates  are  compounds  of  carbon,  hydrogen  and  oxygen  in  which 
the  hydrogen  and  oxygen  are  present  usually  in  the  same  pro- 
portions as  in  water,  hence  the  name  of  the  group.  A  few 
carbohydrates  do  not  conform  to  this  general  statement,  for 
example  Rhamnose  CcHioOj.  Some  substances  not  carbohy- 
drates do  contain  hydrogen  and  oxygen  in  this  proportion,  such 
as  acetic  acid  C2H4O2  and  lactic  acid  CsHgOs.  Thus,  this  char- 
acteristic is  not  distinctive  of  the  group.  Carbohydrates  vary 
considerably  in  their  properties.  They  are  found  both  in  plants 
and  in  animals,  but  chiefly  in  the  former,  in  which  they  form 
a  considerable  part  of  the  structural  frame  work.  In  animals 
they  serve  mainly  as  a  fuel  to  be  oxidized  for  the  production 
of  heat  or  the  performance  of  mechanical  work,  but  they  may 
be  laid  away  as  a  reserve  store  to  be  called  on  in  case  of  need. 

Structure  of  the  Carbohydrates. — The  carbon  atoms  in  the 
carbohydrate  molecule  are  linked  together  in  a  long  chain.  It 
has  been  shown  that  the  molecule  contains  several  hydroxyl 
groups,  and  that  in  the  simpler  members  of  the  group  at  least 

H 

I  I 

there  is  either  an  aldehyde — C  =  0  or  a  ketone  C  =  0  group. 

I  I 

The  remaining  valencies  of  the  carbon  atoms  in  the  chain  hold 
the  hydrogen  atoms  not  accounted  for.  Thus  the  formula 
for  glucose,  one  of  the  most  important  carbohydrates,  has  been 
shown  to  be  as  folloAvs: 

27 


28 


PnYSIOLOGICAL 

CHEMISTRY 

H 

1 

=0 

H- 

1 
-C- 

-OH 

HO- 

-C- 

-H 

H- 

-C- 

1 

-OH 

H- 

1 

-C- 

1 

-OH 

GH^OH 

Glucose 

An  example  of  a  sugar  containing  a  ketone  group  is  fructose, 
which  has  the  following  formula: 

CH^OPI 

I 
C=0 

I 

HO— C— H 

I 
H— C— OH 

I 
H— C— OH 

I 
CH2OH 

Fructose 

Many  of  the  reactions  characteristic  of  the  carbohydrates  de- 
pend upon  the  properties  of  the  aldehyde  or  ketone  groups 
which  they  contain.  In  the  case  of  the  more  complex  carbo- 
hydrates, these  reactive  groupings  are  usually  combined  in  such 
a  way  that  they  do  not  show  their  characteristic  behavior. 

Optical  Activity. — On  inspecting  the  above  formulae  for 
glucose  and  fructose  it  will  be  observed  that  the  hydrogen  and 
hydroxyl  groups  of  the  four  carbon  atoms  occupying  the  middle 
portion  of  the  chain  are  not  arranged  in  a  regular  manner. 


CARBOHYDRATES  29 

This  irregular  arrangement  is  intended  to  indicate  that  there 
is  actually  a  variation  in  the  arrangement  of  these  groups 
around  the  carbon  atoms  in  space.  This  arrangement  is  a 
determining  factor  in  the  property  of  these  substances  known 
as  optical  activity.  Optical  activity  is  the  property  possessed 
by  many  compounds  of  rotating  the  plane  of  polarized  light. 

If  a  ray  of  white  light  is  passed  through  a  crystal  of  Iceland 
spar  it  is  split  into  two  slightly  diverging  rays,  but  this  is  not 
the  only  change  which  is  produced  in  the  ray.  Light  is  due  to 
vibration  of  the  particles  of  the  ether,  the  vibration  being  at 
right  angles  to  the  direction  of  the  ray,  and  in  all  possible 
planes  passing  through  the  path  of  the  ray  as  an  axis.  We 
can  picture  this  perhaps  by  imagining  the  cross-section  of  a 
ray  of  light  to  resemble  the  cross-section  of  an  orange,  only  with 
many  more  planes  of  vibration.  After  passing  through  the 
crystal  of  iceland  spar,  the  light  is  so  altered  that  in  each  of 
the  two  em-erging  rays  vibration  is  taking  place  in  only  one  di- 
rection. To  light  of  this  character  is  given  the  name  plane 
polarized  ligJit.  It  has  been  found  that  optically  active  sub- 
stances have  the  property,  if  such  a  ray  of  light  is  passed 
through  their  solutions,  of  rotating  the  plane  in  which  the 
ether  particles  are  vibrating,  some  substances  rotating  the  plane 
of  vibration  to  the  right,  others  to  the  left.  Not  only  do  these 
compounds  possess  this  property,  but  a  given  substance,  under 
similar  conditions  of  observation  (concentration,  temperature, 
etc.)  always  rotates  the  plane  of  polarized  light  through  the 
same  angle.  This  uniformity  of  behavior  on  the  part  of  each 
optically  active  substance  gives  us  a  most  useful  means  of  de- 
tecting the  presence  of  the  compound  in  question. 

In  order  to  have  a  uniform  standard  for  comparing  observa- 
tions of  optical  activity  it  is  necessary  to  adopt  some  system 
for  reporting  such  data.  A  value  has  been  selected  and  given 
the  name  ''Specific  Rotation,"  which  is  the  rotation  produced 
by  1  gram  of  substance  dissolved  in  1  cubic  centimeter  of  sol- 
vent and  the  rotation  observed  in  a  tube  1  decimeter  in  length. 
This  value  is  designated  by  the  symbol  [  cc  ] . 


30  PHYSIOLOGICAL    CHEMISTRY 

It  is  obvious  that  it  often  will  be  impossible  to  observe  the 
rotation  produced  by  a  substance  under  these  standard  condi- 
tions. Many  substances  are  not  sufficiently  soluble  to  dissolve 
1  gram  in  1  cubic  centimeter  of  solvent.  To  avoid  this  difficulty, 
a  formula  has  been  developed  for  use  under  general  laboratory 
conditions.  Let  cc  be  the  observed  rotation  of  a  solution  under 
question.  Under  standard  conditions  [  oc  ]  =  cc .  Let  g  =  grams 
substance  per  cc.  of  solvent.     If  g  is  not  equal  to  1,  we  can 

easily  find  the  value  of  [  oc  ]  by  dividing  oc  by  g.  [  cc  ]  = '- 

We  must  also  introduce  the  length  of  the  observation  tube,  as 
naturally  a  column  of  liquid  longer  or  shorter  than  the  specified 
1  decimeter  will  give  respectively  a  greater  or  a  smaller  rota- 
tion.   Our  formula  will  now  be  [  oc  1  =-- — - 

l.g 

As  it  seldom  is  convenient  to  work  with  1  cc.  of  solution,  it 
will  be  advisable  to  revise  our  formula  for  use  with  solutions 
calculated  on  the  basis  of  100  cc     Let  c  equal  the  number  of 

grams  substance  in  100  cc.  solvent,  then  g  =  y^  Substituting 

this  value  of  g  in  the  above  equation  we  have 

r        ,         100.  oc 

Now  the  amount  of  rotation  which  a  given  solution  will  pro- 
duce is  influenced  by  various  factors,  among  them  the  tempera- 
ture of  the  solution,  the  color  of  the  light  used  in  the  observa- 
tion, etc.  Unless  there  are  special  reasons  for  other  procedures, 
it  is  customary  to  make  observations  at  20°  C.  and  with  sodium 
light,  corresponding  to  the  D  line  in  the  spectrum.  These  two 
influencing  factors  also  are  included  in  our  formula,  which 
we  now  have  in  its  final  form. 

r        .  20°       100.  cc 


D  l.c 

This  value  is  called  the  specific  rotation  of  a  compound  and 
is  a  constant  for  each  optically  active  substance.  The  specific 
rotations  of  most  of  the  sugars  have  been  determined,  and  may 


CARBOHYDRATES  31 

be  found  in  textbooks  or  books  of  reference.  By  observing  the 
rotation  oc  of  a  solution  of  a  known  sugar  and  substituting  this 
value  in  the  above  equation  it  is  possible  to  calculate  the  amount 
of  the  given  sugar  present.  On  the  other  hand,  if  we  have 
a  solution  containing  a  known  amount  of  an  unknown  sugar, 
it  is  possible  to  calculate  its  specific  rotation,  which  usually  will 
identify  the  substance,  especially  if  used  in  conjunction  with 
other  tests.    For  this  purpose  we  may  solve  our  formula  for  c 

100.  oc 
^  ~'l.  [  oc  ]  20° 
D 
The  actual  observation  of   oc  is  made  with  a  polariscope,  an 
instrument  in  which  light  is  polarized  and  passed  through  the 
solution  to  be  examined.     The  apparatus  is  so  constructed  that 
it  is  possible  to  measure  the  amount  of  rotation  produced  by 
the  solution.     The  accompanying  diagram  indicates  the  struc- 
ture of  a  Laurent  polariscope. 


b     G  d  e  f  g  li  i 

Fig.   1. — Diagram  of  Laurent  Polariscope. 

A.  Source  of  light, — sodium  flame. 

B.  Plate  cut  from  a  crystal  of  potassium  bichromate.     In  place  of  this,  a  flat-sided  cell 

containing  a  solution  of  potassium  bichromate  may  be  used  to  insure  absence  of 
other  than   yellow  light. 

C.  Lens  to  make  the   rays  of  light  parallel. 

D.  Nicol  prism,  called  the  polarizer,  which  polarizes  the  ray. 

E.  Quartz   plate   covering   a   portion   of   the   field   to   produce   half   shadow 

F.  Tube  containing  solution  to  be  studied. 

G.  Second   Nicol   prism   called  the   analyzer. 
H.  Lenses  for  focusing. 

I.     Eye   of  observer. 

The  Nicol  prism  is  a  device  for  polarizing  light,  and  getting 
rid  of  one  of  the  two  rays  into  which  the  original  ray  is  split. 
A  crystal  of  calcite  is  sawed  through  diagonally,  and  the  two 
pieces  stuck  together  with  a  thin  layer  of  Canada  Balsam. 

On  entering  the  prism,  light  is  polarized  and  split  into  two 
diverging  rays.  From  the  diagonal  surface,  one  of  the  two 
polarized  rays  is  reflected  to  the  side,  the  other  passes  on  through 


32  PHYSIOLOGICAL    CHEMISTRY 

the  apparatus.  If  the  tube  /  contains  only  water,  the  polarized 
ray  passes  through  it  without  altering  the  direction  of  its 
vibration.  On  arriving  at  the  second  Nicol  prism  g  the  ray 
will  pass  through  unaltered  provided  the  prism  is  in  a  posi- 
tion corresponding  to  that  of  the  first  prism.  If,  on  the  other 
hand,  the  prism  g  has  been  rotated  to  the  right  or  the  left. so 
that  its  position  no  longer  corresponds  to  that  of  d,  only  a  por- 
tion of  the  ray  will  pass  through,  and  the  intensity  of  the  ray 
reaching  the  eye  at  i  will  be  diminished.  If  the  prism  g  is 
rotated  9'0°  from  the  position  corresponding  to  that  of  d,  no 
light  will  pass  through.  Beyond  this  point  the  illumination  in- 
creases until  at  180°  it  again  is  at  its  maximum.  At  270°  the 
field  again  is  dark. 

Imagine  the  apparatus  set  with  the  two  Nicol  prisms  in  cor- 
responding positions.  Light  will  pass  through  to  the  eye.  Now 
if  a  tube  of  sugar  solution  is  placed  at  /,  the  ray  of  light 
passing  from  d  will  be  rotated  about  its  axis  by  the  sugar  solu- 
tion, which  is  optically  active.  The  result  will  be  that  it  strikes 
g  in  a  position  in  which  it  will  not  pass  through  without  losing 
some  of  its  intensity.  If  we  rotate  prism  g  through  the  same 
angle  through  which  the  ray  of  light  has  been  rotated  by  the 
sugar  solution,  the  ray  again  will  pass  through  at  maximum 
intensity.  By  observing  the  angle  through  which  the  prism 
must  be  rotated  to  bring  this  about,  the  angle  of  rotation  of 
the  ray  caused  by  the  sugar  solution  is  determined. 

In  practice  it  would  be  difficult  to  observe  a  changing  illu- 
minated field  and  select  the  point  at  which  the  illumination  was 
at  its  maximum.  A  mechanism  has  been  devised  to  obviate  this 
difficulty.  This  is  represented  in  the  diagram  by  e  which  is 
a  thin  quartz  plate  covering  one-half  the  visible  field.  This 
plate  so  alters  the  light  passing  through  it  that  when  the  half 
of  the  field  not  covered  by  the  plate  is  at  its  maximum  illu- 
mination, the  half  of  the  field  covered  by  the  plate  is  somewhat 
darker,  and  vice  versa.  By  rotating  the  prism  g  a  point  can 
be  found  intermediate  between  the  two,  at  which  the  two 
halves  of  the  field  are  equally  light.    This  is  taken  then  as  the 


CARBOHYDRATES  33 

starting  (or  zero)  point  of  an  observation.  When  the  sugar 
solution  has  been  introduced  the  prism  g  is  rotated  until  the 
two  halves  of  the  field  again  are  equally  light,  and  the  reading 
taken.  This  will  correspond  to  the  rotation  produced  in  the 
polarization  plane  of  the  ray  by  the  sugar. 

The  readings  are  made  upon  a  circular  scale  which  usually 
is  graduated  in  degrees,  and  provided  with  a  vernier  to  make 
it  possible  to  read  to  minutes. 

The  rotation  produced  by  a  given  substance  varies  with  the 
solvent.  Water  is  the  solvent  most  frequently  used.  If  more 
than  one  optically  active  substance  is  present  in  the  same  solu- 
tion, account  must  be  taken  of  this  fact,  or  one  or  the  other 
removed.  Certain  sugars  when  first  dissolved  in  water  show  a 
much  higher  or  much  lower  rotation  than  after  standing  for 
twenty-four  hours  or  so.  This  is  believed  to  be  due  to  the  fact 
that  these  substances  exist  in  two  forms,  of  which  one  possesses 
much  stronger  rotating  power  than  the  other.  The  two  forms 
pass  into  one  another  spontaneously,  and  reach  an  equilibrium 
in  which  there  is  a  definite  amount  of  each,  the  rotation  pro- 
duced by  this  final  mixture  being  the  resultant  of  the  rotations 
of  the  two  forms  present.  To  this  phenomenon  is  given  the 
name  Mutarotation  (from  the  Latin  word  meaning  "to 
change").  Some  sugars  show  just  twice  the  final  rotation  when 
first  dissolved,  e.g.,  glucose,  and  for  such  cases  the  term  birota- 
tion  may  be  employed.  Half  rotation  is  the  term  employed  for 
those  substances  showing  half  the  final  rotation  when  first  dis- 
solved. Instead  of  allowing  a  solution  to  stand  until  equilibrium 
is  reached,  this  may  be  accomplished  at  once  by  adding  a  small 
amount  of  alkali  to  the  solution. 

The  exact  reason  why  some  compounds  have  the  property  of 
rotating  the  plane  of  polarized  light  is  unknown.  This  property 
has  been  shown  to  depend,  however,  upon  the  presence  of  what 
has  been  called  an  asymmetric  carbon  atom,  which  is  a  carbon 
atom  united  to  four  dissimilar  chemical  groups.  If  the  struc- 
tural formula  for  glucose  is  inspected,  it  will  be  seen  that  in 
the  case  of  four  of  the  carbon  atoms  in  the  chain  each  is  united 


34  PHYSIOLOGICAL    CHEMISTRY 

to  four  different  chemical  groupings.  Each  of  these  four  car- 
bon atoms  is  thus  asymmetric,  and  confers  the  property  of  opti- 
cal activity  upon  the  compound.  Around  each  asymmetric 
atom  either  of  two  groupings  may  exist,  one  of  which  rotates 
the  plane  of  polarized  light  to  the  right  (dextro-rotatory),  the 
other  an  equal  distance  to  the  left  (levo-rotatory).  These  two 
compounds  are  called  optical  isomers.  It  has  been  found  that 
the  number  of  optical  isomers  possible  when  a  compound  con- 
tains more  than  one  asymmetric  carbon  atom,  is  represented 
by  the  value  of  2°  where  n  is  the  number  of  such  carbon  atoms. 
We  will  thus  expect  to  find  2*==16  different  sugars,  all  isomers 
of  glucose.  Twelve  of  these  sixteen  actually  have  been  prepared. 
Classification  of  Carbohydrates. — The  carbohydrates  are 
divided  into  three  great  classes. 

These  are  subdivided  as  indicated  below. 
Monosaccharides 
Bioses 
Trioses 
Tetroses 

Pentoses — arabinose,  xylose,  ribose 
Hexoses — glucose,  fructose,  galactose,  mannose 
Heptoses 
Octoses 
Nonoses 

Disaccharides 

Saccharose  (sucrose,  or  cane  sugar) 

Maltose  (malt  sugar) 

Lactose  (milk  sugar) 
Polysaccharides 

Dextrins 

Starches 

Glycogen 

Inulin 

Cellulose 

Gums  and  Mucilages. 


CARBOHYDRATES  35 

The  monosaccharides  are  sometimes  called  the  simple  sugars. 
The  disaccharides  are  so  called  because  they  are  formed  by  the 
union  of  two  molecules  of  monosaccharide,  with  elimination  of 
water.  Polysaccharides  are  composed  of  several  molecules  of 
monosaccharide  united  in  a  similar  manner.  The  monosac- 
charides are  subdivided  into  groups  according  to  the  number  of 
carbon  atoms  in  the  molecule,  thus  the  bioses  are  sugars  hav- 
ing only  two  carbon  atoms  in  each  molecule,  the  trioses,  three, 
etc.  The  first  six  of  these  groups  are  found  in  nature.  The 
octoses  and  nonoses  have  been  built  up  in  the  laboratory.  Of 
the  monosaccharides,  the  pentoses  and  hexoses  are  the  most  im- 
portant. With  the  exception  of  the  bioses,  there  are  two 
classes  of  sugars  in  each  group, — aldehyde  sugars  and  ketone 
sugars.  Of  the  biologically  important  sugars  all  are  aldehyde 
sugars,  or  aldoses,  with  the  exception  of  fructose,  which  is  a 
ketone  sugar  or  ketose.  The  individual  carbohydrates  will  be 
discussed  later. 

Origin  and  Synthesis. — The  ultimate  dependence  of  the 
animal  world  upon  plants  is  well  illustrated  by  the  carbohy- 
drates. These  compounds  which  are  an  important  fuel  for 
the  body  are  obtained  from  plants,  in  which  they  are  built  up 
from  the  very  simple  substances  carbon  dioxide  and  water.  The 
exact  mechanism  by  which  the  plant  brings  about  this  important 
synthesis  is  a  matter  of  some  uncertainty,  but  it  is  probable  that 
the  carbon  dioxide  from  the  air  is  first  reduced  to  formaldehyde. 
Several  molecules  of  formaldehyde  then  condense  to  form  a 
carbohydrate.     The  reaction  might  be  represented  as  follows: 

CO^+H^O-^H-CHO+Oa 
6  HCHO^CeH.A 

The  energy  for  this  synthesis  is  derived  from  sunlight  by  the 
agency  of  chlorophyl,  the  green  coloring  matter  of  plants. 
The  hexose  so  formed  may  then  be  built  up  into  more  complex 
substances  such  as  starch,  which  is  laid  away  as  reserve  food 
in  the  seeds,  tubers  and  other  parts  of  the  plant.  Sunlight  is 
not  necessary  for  the  second  part  of  this  process,  as  starch  can 
be  built  up  in  the  roots  and  tubers,  wdiich  are  underground.    By 


36  PHYSIOLOGICAL    CHEMISTRY 

variations  of  this  general  process  the  plant  undoubtedly  can 
build  up  a  large  number  of  other  compounds  of  the  most  diverse 
nature. 

The  animal  body  has  much  more  limited  powers  of  synthesiz- 
ing carbohydrates,  although  we  now  know  that  it  is  capable 
of  doing  much  more  in  this  respect  than  was  once  thought.  The 
polysaccharide  glycogen  is  regularly  built  up  in  the  animal 
body  from  monosaccharides,  and  it  has  been  shown  in  diseases 
where  carbohydrates  are  lost  from  the  body  in  the  urine,  that 
certain  compounds  other  than  carbohydrates  apparently  can  be 
converted  into  sugar  in  the  body. 

There  are  various  methods  for  synthesizing  carbohydrates  in 
the  laboratory.  One  of  these  suggests  the  synthesis  in  plants. 
If  a  solution  of  formaldehyde  is  made  slightly  alkaline,  a  con- 
densation takes  place,  and  the  liquid  will  be  found  to  contain 
a  hexose,  acrose. 

A  method  which  has  proved  very  useful  in  studying  the 
carbohydrates  is  known  as  the  cyanhydrin  synthesis.  This 
serves  to  lengthen  the  carbon  chain  by  one  carbon  atom.  Start- 
ing from  a  pentose,  a  hexose  may  be  prepared,  from  a  hexose 
a  heptose,  and  so  on.    The  steps  of  the  synthesis  are  as  follows : 

CH^OH— CHOH— CHOH— CHOH— CHO+HCN^ 
CH,OH— CHOH— CHOH— CHOH— CHOH— CN 

This  nitril,  containing  six  carbon  atoms  is  easily  saponified. 

HOH 
CH.OH— CHOH— CHOH— CHOH— CHOH— CN+HOH^ 

HOH 

CH.OH— CHOH— CHOH— CHOH— CHOH— COOH+ 
NH3+H,0 

Converting  this  into  its  lactone  by  the  action  of  acid  we  get: 

CH2OH— CHOH— CH— CHOH— CHOH— C=0+H20 

I o 1 

On  reducing  this  with  sodium  amalgam  the  compound  takes 


CARBOHYDRATES  37 

up  2H  forming  an  aldehyde,  a  hexose,   which  thus  has  been 
built  up  from  a  pentose. 

CH^OH— CHOH— CHOH— CHOH— CHOH— CHO 

• 

The  carbohydrates  also  may  be  "built  down"  step  by  step  in 
the  laboratory.  This  method  also  has  been  of  value  in  study- 
ing their  constitution.  If  treated  with  hydrogen  peroxide  in 
the  presence  of  a  ferric  salt  as  catalyzer,  the  elements  of  formic 
acid  are  split  off,  leaving  a  carbohydrate  with  one  less  carbon 
atom.  Carbohydrates  also  may  be  built  down  by  electrolysis 
or  by  way  of  their  oximes. 

Interconversion  of  Carbohydrates. — Since  the  different  mono- 
saccharides are  closely  related  compounds,  differing  only  in  the 
arrangement  of  -their  hydrogen  and  hydroxyl  groups  around  the 
carbon  atoms,  it  is  not  surprising  to  learn  that  certain  of  them 
may  very  easily  be  converted  into  one  another.  Thus  if  a  solu- 
tion of  glucose  is  made  slightly  alkaline  and  allowed  to  stand 
for  some  time,  it  will  be  found  to  contain  not  glucose  alone, 
but  also  fructose  and  mannose.  A  portion  of  the  glucose  is 
transformed  into  the  other  two  sugars.  It  is  immaterial  which 
of  the  three  sugars  is  taken  to  start  with, — the  result  will  be 
the  same  and  all  three  will  be  found  in  the  solution.  The  inter- 
conversion of  the  simple  sugars  is  of  interest  physiologically, 
for  in  the  body  such  transformations  are  known  to  take  place. 
The  lactose  of  milk  is  produced  in  the  mammary  gland  from 
glucose  in  the  blood.  Lactose  is  made  up  of  equal  parts  of  glu- 
cose and  galactose.  A  portion  of  the  glucose  from  the  blood 
thus  must  be  transformed  into  galactose  in  the  gland.  A  second 
instance  of  a  similar  transformation  is  the  formation  of  the 
polysaccharide  glycogen  which  is  stored  up  in  the  liver  and 
muscles  as  a  reserve  material.  On  hydrolysis  glycogen  yields 
always  glucose.  It  is  well  known,  however,  that  glycogen  will 
be  deposited  in  the  body  if  an  animal  is  fed  fructose  or  various 
other  sugars.  The  glycogen  formed  under  these  circumstances 
also  yields  glucose  on  hydrolysis,  indicating  the  transformation 
of  the  fructose,  etc.,  into  glucose. 


38  PHYSIOLOGICAL    CHEMISTRY 

Combination  of  Carbohydrates  With  One  Another,  and  With 
Other  Substances. — The  carbohydrates  have  the  property  of 
combining  with  various  substances  including  themselves.  The 
union  of  two  molecules  of  monosaccliaride  forms  the  disac- 
charides,  of  many  molecules  of  monosaccharide,  the  polysac- 
charides. Compounds  of  monosaccharides  with  other  classes  of 
materials  are  variously  named,  and  include  substances  of  great 
biological  interest  and  importance.  Examples  are  the  glucosides, 
— compounds  of  glucose  or  one  of  its  derivatives,  among  which 
are  many  of  the  drugs  used  in  medicine,  and  some  of  the  constit- 
uents of  brain  and  nerve  tissue.  The  term  "Glucosides"  is 
sometimes  extended  to  include  similar  compounds  which  yield 
other  sugars,  e.g.,  galactose. 

Behavior  With  Strong  Alkalies. — The  action- of  weak  alkali 
upon  the  carbohydrates  already  has  been  discussed  under  the 
heading  interconversion  of  carbohydrates.  A  rearrangement  of 
groups  in  the  molecule  is  observed.  The  action  of  strong  alkali 
is  much  more  vigorous  and  far  reaching  and  the  nature  of  the 
products  formed  depends  upon  the  experimental  conditions. 
The  alkali  undoubtedly  combines  with  the  sugar  at  first.  There 
follows  loss  of  water  from  groups  around  neighboring  carbon 
atoms,  and  a  double  bond  is  formed.  The  carbon  chain  is  then 
broken,  and  two  compounds  are  formed,  each  with  a  smaller 
number  of  carbon  atoms  than  the  original  substance.  The  nature 
of  these  fragments  is  only  imperfectly  understood,  but  it  may 
be  inferred  from  the  final  products  of  the  reaction.  In  case  oxy- 
gen is  supplied  plentifully,  the  fragments  are  oxidized  to  the 
corresponding  acids.  Of  these  a  long  list  may  be  obtained  vary- 
ing in  complexity  from  formic  H  .  COOH  and  oxalic  acids 

COOH  to  acids  having 

I 
COOH 

chains  as  long  or  only  slightly  shorter  than  the  chain  in  the 
original  substance.  Evidently  the  breaking  up  into  fragments 
may  take  place  at  various  points  in  the  chain,  giving  compounds 


CARBOHYDRATES  39 

containing  one,  two,  three,  four,  or  five  carbon  atoms.  The 
chain  also  may  remain  unbroken. 

This  process  is  of  biological  interest  for  probably  it  has  many 
points  of  similarity  with  the  breaking  down  of  carbohydrates  in 
the  body.  Of  course  the  body  tissues  are  not  strongly  alkaline, 
so  that  the  agent  bringing  about  the  change  must  be  some  other 
substance.  In  the  body  the  fragments  thus  produced  may  be 
used  to  construct  new  substances,  or  they  may  be  further  broken 
down  and  oxidized  to  carbon  dioxide. 

In  case  the  strong  alkali  acts  upon  glucose  when  the  oxygen 
supply  is  limited,  as  would  be  the  case  if  no  air  were  bubbled 
through  the  liquid,  the  reactive  fragments  produced  will  com- 
bine with  one  another,  forming  complex  brown  substances  of  a 
resinous  nature,  a  mixture  of  which  is  known  as  caramel.  Pos- 
sibly this  process  is  analogous  to  the  building  up  of  materials 
from  carbohydrate  fragments  in  the  interior  of  the  body  cells, 
although  in  this  latter  case  the  conditions  of  synthesis  are  care- 
fully controlled  by  agents  in  the  cell  so  that  particular  sub- 
stances result  which  are  required  by  the  cell. 

Behavior  With  Acids. — On  boiling  with  dilute  acids  the  com- 
plex carbohydrates  take  up  water  and  are  split  into  simpler 
substances,  ultimately  the  monosaccharides.  If  the  monosac- 
charides are  boiled  with  strong  hydrochloric  acid  they  are  de- 
composed and  yield  a  variety  of  products.  The  pentoses  lose 
water  and  form  furfurol. 

lEI    H      H     El     H 

H      C  — C  — C  — C  — 0  =  0  HC  —    OH 

/                          I  -^    II             II              +3H,0 

^  HO           C— CHO 


The  formation  of  this  compound  serves  to  identify  the  pen- 
toses, since  it  forms  with  various  phenols  colored  substances 
which  may  be  identified  easily.  The  hexoses,  on  boiling  with 
concentrated  hydrochloric  acid  yield  among  other  things  oxy- 
methyl  furfurol,  but  in  much  larger  quantities  levulinic  acid 


40  PHYSIOLOGICAL    CHEMISTRY 

CH3 .  C— CH2 .  CH2 .  COOH 

II 

0 

Oxidation  of  Carbohydrates. — Oxidation  Tests. — Since  the 
simple  carbohydrates  contain  either  an  aldehyde  or  a  ketone 
group  they  are  easily  oxidized,  even  by  mild  oxidizing  agents. 
The  products  of  mild  oxidation  are  acids  having  the  same  num- 
ber of  parbon  atoms  as  the  original  material.  If  the  oxidation  is 
more  vigorous  the  molecule  may  break  into  fragments  as  was  de- 
scribed under  the  action  of  alkalies.  Some  of  the  most  important 
carbohydrate  tests  depend  upon  oxidation  processes.  Among 
the  oxidation  tests  are  the  Fehling,  Almen-Nylander,  and  Bar- 
foed  tests. 

Feliling  Test. — If  solutions  of  copper  sulphate  and  sodium 
hydroxide  are  mixed,  a  light  blue  or  whitish  precipitate  is 
formed.     This  is  cupric  hydroxide 

CuS04-h2  NaOH-^Cu(OH).+Na2S04 

If  this  mixture  is  boiled,  the  precipitate  is  converted  into 
black  cupric  oxide. 

Cu(OH)o->CuO+H20. 
If  a  sugar  is  present  in  the  solution,  however,  the  copper  is 
reduced  by  the  sugar  which  is  converted  into  an  acid.  If  a  sugar 
is  added  to  a  mixture  of  copper  sulphate  and  sodium  hydrate  the 
liquid  will  turn  a  very  deep  blue,  and  a  much  smaller  precipitate 
will  form  as  most  of  the  Cu(0II)2  is  held  in  solution  by  com- 
bining with  the  sugar.  If  the  mixture  is  allowed  to  stand,  or 
if  it  is  boiled,  the  color  becomes  perhaps  yellow  at  first,  and 
ultimately  a  red  precipitate  forms.  The  sugar  has  reduced  the 
cupric  hydroxide  to  yellow  cuprous  hydroxide,  which,  on  boiling, 
decomposes  into  the  red  cuprous  oxide. 

2  Cu(OH)2+C5HiiO,.CH0^2  CuOH-f 

C5H11O5 .  COOH+H2O 
2  CuOH^Cu^O+H^O 

As  a  matter  of  fact,  the  sugar  undoubtedly  undergoes  further 
change,  as  we  already  have  seen  in  considering  the  action  of  al- 


CARBOHYDEATES  41 

kalies  on  the  carbohydrates,  but  the  above  equations  serve  to 
show  the  nature  of  the  part  played  by  the  copper  compound  in 
the  reaction. 

The  above  test  would  work  very  well  if  the  solution  to  be 
tested  contained  much  sugar.  If  this  were  not  the  case,  how- 
ever, much  black  cupric  oxide  would  be  formed  which  might 
easily  obscure  any  small  amount  of  red  cuprous  oxide  resulting 
from  the  reducing  action  of  a  small  amount  of  sugar.  Accord- 
ingly it  is  more  satisfactory  to  use  Fehling  's  solution  for  the  test. 
This  is  made  up  in  two  parts,  A  and  B,  which  are  mixed  in  equal 
quantities  immediately  before  using.  A  contains  copper  sul- 
phate; B  contains  sodium  hydrate  and  sodium  potassium  tar- 
trate. On  mixing  these  two  solutions  a  deep  blue  liquid  results. 
The  two  solutions  are  kept  separate,  as  otherwise  the  tartrate 
will  slowly  reduce  the  copper.  The  advantage  in  Fehling 's  rea- 
gent lies  in  the  fact  that  the  sodium  potassium  tartrate  unites 
with  the  cupric  hydroxide  to  form  a  complex  ion ;  thus  the  cupric 
hydroxide  does  not  precipitate  and  does  not  decompose  into  the 
black  cupric  oxide.  On  boiling,  the  liquid  remains  clear  and 
blue.  The  combined  cupric  hydroxide  is  in  equilibrium  with  a 
xevy  small  amount  of  this  compound  in  solution  so  that  as  fast 
as  the  free  cupric  hydroxide  is  reduced  by  the  sugar  solution, 
more  of  the  copper-hydrate-tartrate  compound  dissociates.  This 
complex  compound  thus  furnishes  a  ready  supply  of  copper 
hydroxide,  and  if  sufficient  sugar  is  present,  all  of  the  copper 
will  be  reduced.  At  this  point  the  blue  color  will  have  disap- 
peared from  the  liquid.  The  following  equation  illustrates  the 
formation  of  the  complex  compound : 

COO  coo 

I  I 

CHOH+2Cu(OH)2^  CHO— CUOH+2H2O 

I  I 

CHOH  CHO— CuOH 

i  I 

COO  coo 


42  PHYSIOLOGICAL    CHEMISTEY 

The  Fehling  test  will  detect  0.1%  glucose  in  a  solution. 

Barfoed's  Test.— This  test  also  depends  upon  the  reduction  of 
a  copper  salt  (copper  acetate)  by  the  sugar.  It  is  performed, 
however,  in  acid  solution.  Barfoed's  reagent  contains  copper 
acetate  and  acetic  acid.  The  reducing  action  of  the  carbohy- 
drates is  very  much  less  in  acid  than  in  alkaline  solution.  All 
of  the  reducing  carbohydrates  will  reduce  Barfoed's  reagent  if 
boiled  a  sufiEicient  length  of  time,  producing  red  cuprous  oxide 
as  in  the  Fehling  test.  The  monosaccharides,  however,  reduce 
Barfoed's  solution  faster  than  do  the  disaccharides  in  equal 
concentration,  so  that  if  properly  used,  this  test  may  serve  to 
distinguish  between  monosaccharides  and  disaccharides.  But  a 
concentrated  maltose  solution  will  reduce  Barfoed's  reagent 
more  rapidly  than  a  weak  glucose  solution,  so  that  this  fact 
should  be  borne  in  mind  or  erroneous  conclusions  may  result. 

Almen-Nylander  Test. — ^Nylander's  reagent  contains  bismuth 
subnitrate,  sodium  potassium  tartrate  and  potassium  hydroxide. 
The  part  played  by  each  constituent  is  similar  to  that  in  Feh- 
ling's  solution.  The  bismuth  subnitrate  is  reduced  to  black, 
metallic  bismuth.     The  equations  follow: 

Bi  ( OH )  ^NOg-f  KOH^Bi  ( OH )  3+KNO3 

2  Bi ( OH) 3+Sugar-^Bi2+3H20+  ( Sugar+30) 

Certain  substances  which  interfere  with  Fehling 's  test,  have 
no  disturbing  influence  on  the  Nylander  reaction  (uric  acid, 
creatinine)  so  that  this  test  is  useful  occasionally  when  the  Feh- 
ling test  is  of  questionable  value.  A  solution  containing  0.08% 
glucose  will  give  a  positive  Nylander  test. 

Haines'  solution  differs  from  Fehling 's  solution  in  contain- 
ing glycerine  in  place  of  sodium  potassium  tartrate.  Its  deli- 
cacy is  about  equal  to  that  of  the  Fehling  test. 

Reduction  of  Carbohydrates. — ^By  the  action  of  reducing 
agents  carbohydrates  may  be  converted  into  alcohols,  or  on  fur- 
ther reduction  they  may  give  rise  to  compounds  of  the  nature 
of  fatty  acids.  Such  transformations  apparently  occur  in  the 
cells    of    the    body,    for    it    is    a    well    known    fact    that    a 


CARBOHYDRATES  *  43 

carbohydrate  diet  is  "fattening."  Possibly  the  carbohydrate 
molecules  are  both  split  into  fragments  and  reduced  or  dehy- 
drated, and  then  recombined  to  form  compounds  with  longer 
chains, — fatty  acids.  The  exact  mechanism  of  the  process  is 
still  unknown.  It  is  quite  probable  that  carbohydrates  may  give 
up  their  oxygen  to  cells  or  microorganisms  under  conditions 
where  vital  activities  are  going  on  in  the  absence  of  atmospheric 
oxygen.     This  process  is  called  anaerobic  respiration. 

Formation  of  Osazones. — Monosaccharides  and  many  of  the 
disaecharides  combine  with  phenylhydrazine  to  form  osazones. 
These  are  yellow  compounds  which  crystallize  in  needles.  The 
crystals  often  group  together  with  points  at  a  common  center, 
thus  forming  rosettes,  or  fans,  or  sheaves  like  grain  sheaves.  The 
different  osazones  have  slightly  differing  crystal  forms,  but  they 
are  best  recognized  by  their  melting  points;  identifying  an 
osazone  serves  to  identify  the  sugar  from  which  it  was  formed. 
Glucose  and  fructose  form  the  same  osazone,  since  the  struc- 
ture of  these  two  sugars  differs  only  around  the  carbon  atoms 
to  which  the  phenylhydrazine  molecules  become  attached.  This 
test  thus  will  not  distinguish  between  these  two  sugars.  Sac- 
charose, for  a  reason  to  be  seen  later,  does  not  form  an  osa- 
zone, nor  do  the  polysaccharides. 

The  reaction  takes  place  in  three  stages  as  follows: 

CH2OH  CHoOH 

I  i 

CH  OH  (CH  0H)3 

CH  OH  -^       CH  OH 

I  I 

CH  OH  CH  =N  NH  CJI,  +H2O 

CH  OH 

CHO       +H2N     NH      CgHg 

The  product  is  a  hydrazone.  A  second  molecule  of  phenylhy- 
drazine then  removes  two  H  atoms  from  the  group  next  the  end 
carbon  atom,  and  is  converted  into  aniline  and  ammonia. 


44  PHYSIOLOGICAL   CHEMISTRY 

CH2OH  CH2OH  +H2NCeH54-NH3 

I  I 

(CH  0H)3  -^         (CH0H)3 

I  I 

CHOH +H2NNHC6H5  C=0 

I  I 

CH=N  NH  CeHg  CH=N  NH  CeH^ 

A  third  molecule  of  phenyl  hydrazine  now  reacts,  forming 
the  osazone. 

CH,OH  CH2OH 

(CH0H)3+  H^N  NH  CeH^  (CH0H)3  +  H,0 

I  I 

C=0  C=N  NH  CeH. 

I  I 

CH=N  NHCeHg  C  =N  NH  CgH, 

H 

In  place  of  phenyl  hydrazine,  its  hydrochloride  often  is  used, 
as  this  compound  is  more  soluble  and  more  stable  than  the 
free  base. 

The  Molisch  Test. — If  to  a  solution  containing  a  carbohy- 
drate a  few  drops  of  15%  alcoholic  oc  naphthol  are  added,  and 
concentrated  sulphuric  acid  carefully  poured  down  the  side  of 
the  test  tube  so  that  it  will  form  a  layer  at  the  bottom  of  the 
tube,  a  violet  or  reddish  ring  will  form  at  the  juncture  of  the 
two  liquids.  This  test  will  detect  not  only  free  carbohydrate, 
but  also  carbohydrate  in  combination  with  other  substances. 
The  reaction  is  so  delicate,  however,  that  it  will  be  given  by  very 
small  traces  of  carbohydrate  material,  even  fibers  of  filter  paper 
(cellulose)  so  that  as  a  general  test  it  is  more  valuable  in  a 
negative  than  in  a  positve  result.  A  negative  Molisch  reaction 
is  good  evidence  that  carbohydrates  are  absent,  whereas  a  posi- 
tive test  may  be  due  to  the  presence  of  filter  paper  or  other 
casual  impurities.  As  little  as  0.2  mg.  filter  paper  is  said  to 
give  a  strong  reaction. 


CARBOHYDRATES  45 

Fermentation — Enzymes 

Under  the  influence  of  certain  microorganisms  the  carbohy- 
drates undergo  a  process  known  as  fermentation,  which  consists 
in  the  breaking  up  of  the  carbohydrate  molecule  to  form  a 
variety  of  simpler  compounds.  The  nature  of  the  products  de- 
pends upon  the  character  of  the  particular  organism  respon- 
sible for  the  decomposition.  Thus  the  carbohydrate  may  form 
carbon  dioxide  and  alcohol,  a  process  known  as  alcoholic  fer- 
mentation. Or  it  may  form  lactic  acid,  butyric  acid,  or  still 
other  substances.  In  alcoholic  fermentation  we  may  represent 
the  process  as  follows: 

CsH,,0e->2  C^HgOH-f  2CO2 

but  in  reality  there  are  intermediate  steps  the  character  of 
which  is  but  imperfectly  understood.  The  changes  brought 
about  in  the  sugar  in  these  reactions  are  due  to  the  fact  that  the 
microorganisms  involved  contain  or  secrete  compounds  which 
have  the  power  of  breaking  down  the  sugar.  We  know  a  great 
many  of  these  substances,  and  their  activities  are  by  no  means 
limited  to  the  breaking  down  of  sugars.  To  them  has  been  given 
the  name  of  enzymes  and  different  members  of  this  class  of  sub- 
stances have  the  power  of  bringing  about  the  most  widely  vary- 
ing chemical  reactions,  both  breaking  dow^n  substances  and 
building  them  up. 

Enzymes  have  been  the  object  of  much  study  in  recent  years, 
but  as  yet  very  little  is  known  of  their  chemical  constitution. 
There  is  some  evidence  indicating  that  perhaps  some  of  the 
members  of  the  group  may  be,  or  may  closely  resemble  proteins, 
whereas  others  appear  to  be  carbohydrates.  It  is  altogether 
probable  that  they  will  be  found  to  vary  much  in  their  chemical 
nature,  just  as  they  vary  in  their  chemical  activities. 

Although  little  is  known  of  the  chemical  constitution  of  the 
enzymes,  many  important  facts  are  known  concerning  their 
properties  and  the  conditions  governing  their  activities.  One 
of  the  striking  facts  of  enzyme  action  is  that  these  substances  do 
not  appear  combined  with  the  final  products  which  they  produce. 


46  PHYSIOLOGICAL   CHEMISTRY 

They  are  responsible  for  the  breaking  down  or  building  up  of 
many  classes  of  compounds,  but  apparently  they  only  change 
the  speed  of  reactions  which,  if  given  sufficient  time,  would  go 
on  of  themselves.  Substances  which  shown  this  behavior  are 
spoken  of  as  catalytic  agents,  and  are  said  to  act  by  catalysis. 
Enzymes  have  been  defined  as  substances  produced  by  living 
cells,  which  act  by  catalysis. 

It  was  formerly  believed  that  the  enzyme  of  yeast  which 
breaks  down  sugar  into  alcohol  and  carbon  dioxide  acted  only 
within  the  living  cell,  and  that  the  vital  activities  of  the  cell 
were  intimately  connected  with  its  action.  Organisms  of  the 
type  of  yeast  were  called  ferments.  Another  class  of  reactions 
was  known  to  be  independent  of  the  cell  by  which  the  active 
principle  was  produced;  substances  responsible  for  such  reac- 
tions were  called  unorganized  ferments.  Among  them  were 
the  various  digestive  enzymes.  In  1897  this  fallacy  was  cor- 
rected by  Buchner,  who  ground  up  yeast  cells  with  sharp  sand, 
thus  tearing  the  cells  and  allowing  the  cell  fluids  to  escape. 
The  entire  mixture  of  sand  and  broken  cells  was  then  pressed 
in  a  powerful  press.  A  small  quantity  of  liquid  was  obtained 
and  was  filtered  through  a  porous  porcelain  filter  which  held 
back  any  fragments  of  cells.  The  clear  liquid  was  found  to 
decompose  sugar  in  the  same  way  as  the  original  yeast  cells 
had  done.  The  activities  of  the  enzyme  were  thus  in  no  way 
dependent  upon  the  vital  processes  going  on  in  the  cell.  It  is 
not  to  be  supposed  from  this  that  the  enzyme  activities  are  of 
no  value  to  the  cell.  Quite  the  contrary  is  the  case,  for  undoubt- 
edly the  greater  number  of  the  chemical  reactions  which  go  on 
in  the  cell,  and  which  to  a  large  extent  make  up  the  sum  total 
of  what  we  call  its  vital  activities,  depend  upon  simple  chemical 
reactions  which  are  directed  or  controlled  by  enzymes.  We 
still  know  certain  types  of  enzymes  which  thus  far  have  not 
been  isolated  from  the  cells  in  active  form,  but  possibly  this 
will  be  accomplished  in  the  course  of  time. 

Nomenclature  and  Classification. — The  nomenclature  of  the 
enzymes  is  somewhat  irregular,  as  several  of  the  substances 
were  named  before  any  regular  plan  had  been  adopted.     The 


CARBOHYDRATES  47 

ending  "ase"  is  now  employed  to  indicate  that  a  substance  is 
an  enzyme,  and  the  remainder  of  the  word  usually  indicates 
either  the  substance  upon  which  the  enzyme  acts,  the  nature  of 
the  reaction  it  brings  about,  or  some  other  property  of  the  com- 
pound. The  substance  upon  which  the  enzyme  acts  is  called 
the  substrate.  The  classification  of  the  enzymes  is  only  pro- 
visional until  a  better  one  is  possible.  They  are  grouped  accord- 
ing to  the  substances  acted  upon,  or  the  character  of  the  reac- 
tion induced,  thus  there  are  proteases  (act  on  proteins),  lipases 
(act  on  fats),  amylases  (act  on  starch  and  amylum),  lactase 
(on  lactose),  maltase  (on  maltose),  oxidases,  reductases,  etc. 
Of  the  older  names  we  have  pepsin,  remain,  trypsin,  zymase 
(found  in  yeast),  etc. 

Specific  Nature. — As  may  be  inferred  from  the  above  state- 
ments, the  enzymes  act  only  upon  particular  substances  or 
classes  of  substances,  and  in  general  an  enzyme  which  acts  upon 
one  compound  will  not  act  upon  any  other.  The  enzymes  are 
thus  said  to  be  specific  in  their  action, — that  is,  each  one  acts 
only  on  a  particular  kind  of  material,  or  brings  about  one  parti- 
cular kind  of  chemical  action.  Emil  Fischer,  one  of  the  most 
brilliant  chemists  of  all  times,  has  likened  this  characteristic  to 
the  fitting  of  a  key  with  its  lock.  As  a  matter  of  fact,  the  en- 
zymes are  believed  to  fit  onto  the  substances  they  act  upon,  form- 
ing temporary  compounds  which  quickly  break  down  again. 

Influence  of  Temperature. — One  of  the  characteristics  of  the 
enzymes  is  their  extreme  sensitiveness  to  temperature.  The 
temperature  of  the  solution  in  which  an  enzyme  is  acting  will 
be  found  to  influence  greatly  the  speed  of  the  action.  The  tem- 
perature at  which  different  enzymes  will  act  best  is  known  as 
their  optimum  temperature,  and  for  the  enzymes  in  the  body 
this  is  in  the  neighborhood  of  37°-40°  C,  in  other  words  about 
body  temperature.  The  enzymes  in  some  of  the  cold-blooded 
animals,  however,  whose  body  temperature  varies  with  that  of 
their  surroundings,  act  well  at  temperatures  much  lower  than 
this,  and  it  is  obvious  that  enzymes  in  plant  cells  must  work  well 
at  temperatures  much  below  that  of  the  bodj^  If  a  solution 
containing  an  enzyme  is  heated  to  60° -80°  the  enzyme  is  de- 


48  PHYSIOLOGICAL    CHEMISTRY 

stroyed,  and  cooling  the  solution  will  not  restore  its  activity. 
On  the  other  hand,  enzymes  in  general  can  be  exposed  to  tem- 
peratures near  the  freezing  point.  Their  activity  is  retarded  but 
will  return  if  the  solution  is  warmed. 

Effect  of  Chemical  Reaction, — Enzymes  are  also  very  sensi- 
tive to  chemical  reaction.  They  are  destroyed  by  strong  acids 
or  alkalies.  The  most  favorable  reaction  differs  with  different 
enzymes,  some  acting  best  in  weak  acid,  others  in  weak  alkaline 
solution.  Certain  of  the  enzymes  which  act  in  weak  acid  solu- 
tion are  destroyed  by  making  the  solution  even  faintly  alkaline, 
and  the  converse  case  is  also  true.  Some,  on  the  other  hand, 
will  stand  considerable  variation  in  this  respect,  acting  in  weak 
acid,  in  neutral  or  in  weak  alkaline  solution. 

Reversibility. — Some  of  the  enzymes  have  the  power  of 
causing  a  chemical  reaction  to  go  either  way, — -that  is,  of  de- 
composing a  compound,  or  under  proper  conditions  of  building 
up  the  same  compound  from  its  decomposition  products.  Cer- 
tain of  the  lipases  are  notable  examples.  The  property  is  spoken 
of  as  reversibility,  and  these  enzymes  are  said  to  be  reversible  in 
their  action. 

Active  and  Inactive  Form. — In  the  form  in  which  they  are 
secreted  by  cells,  some  of  the  enzymes  are  inactive,  and  become 
capable  of  exerting  their  customary  activity  only  after  they  have 
been  acted  on  by  some  other  substance.  The  enzymes  are  thus 
said  to  exist  in  inactive  and  active  forms. 

Action  Retarded  by  Products, — The  activity  of  an  enzyme  is 
often  retarded  by  the  products  which  it  produces  from  the  sub- 
strate. In  life  (in  vivo)  these  products  usually  are  removed,  as 
in  absorption  of  the  digestion  products  from  the  intestine.  In 
a  laboratory  experiment  in  a  test  tube  or  beaker,  this  may  influ- 
ence the  extent  of  digestion  by  an  enzyme  to  a  considerable  ex- 
tent. This  fact  may  be  summed  up  by  saying  that  enzyme  ac- 
tion often  is  incomplete  in  vitro  (in  glass). 

Progressive  Action, — The  enzymes  are  often  said  to  be  pro- 
gressive in  their  action.  This  is  only  a  special  case  of  their 
specific  nature.     Many  reactions  depend  upon  more  than  one 


CARBOHYDRATES  49 

enzyme  for  completion,  the  reaction  taking  place  in  successive 
stages,  each  one  of  which  is  brought  about  by  a  different  enzyme. 
Thus  the  breakdown  of  starch  into  glucose  requires  at  least  two 
and  possibly  more  enzymes.  The  first  breaks  down  the  starch 
through  various  stages  to  maltose,  the  second  breaks  the  maltose 
into  glucose. 

Summary. — In  summary  it  may  be  said  that  enzymes  are  sub- 
stances of  unknown  chemical  constitution,  which  act  as  catalytic 
agents  affecting  a  large  variety  of  chemical  reactions.  They  are 
specific  in  their  action,  they  are  sensitive  to  changes  in  reaction 
and  temperature,  being  destroyed  if  heated  to  60°-80°  C.  Some 
are  reversible  in  their  action,  they  are  often  secreted  in  inactive 
form  and  become  active  only  on  coming  in  contact  with  some 
other  substance,  their  action  is  often  incomplete  in  vitro,  and 
frequently  is  progressive  in  its  character. 

Individual  Groups  of  Carbohydrates. 

Pentoses. — The  pentoses  are  found  in  both  plants  and 
animals,  usually  combined  with  other  substances  as  in  nucleo- 
proteins  or  in  the  form  of  polysaccharides  made  up  of  many 
molecules  of  pentose.  They  are  obtained  by  the  hydrolysis  of 
these  compounds.  A  pentose,  probably  arabinose,  has  been  found 
in  the  urine.  This  condition  is  known  as  pentosuria.  Pentoses 
may  be  either  aldoses  or  ketoses.  They  reduce  Fehling's  and 
other  similar  solutions,  and  give  osazones  with  phenylhydrazine. 
They  usually  do  not  ferment,  however.  The  pentoses  are 
utilized  by  herbivorous  animals  but  the  extent  to  which  they 
may  be  utilized  by  man  seems  to  be  more  limited,  although  the 
subject  is  still  a  matter  of  some  uncertainty.  Pentoses  may  be 
distinguished  from  hexoses  by  their  osazones,  by  their  failure  to 
ferment  readily,  arid  also  by  certain  color  reactions  among  which 
are  the  orcin  and  phloroglucin  tests.  If  a  pentose  is  heated  with 
concentrated  hydrochloric  acid  and  a  little  orcin  or  phloroglucin, 
a  distinct  color  change  results.  With  orcin  the  color  is  first  vio- 
let, then  blue,  red,  and  finally  green,  and  a  bluish  green  pre- 
cipitate forms.     With  phloroglucin,  the  color  is  red.     As  other 


50  PHYSIOLOGICAL   CHEMISTRY 

substances  will  give  similar  colors,  it  is  necessary  to  confirm  the 
result  by  observing  the  absorption  spectrum  of  the  colored  sub- 
stance after  it  has  been  dissolved  out  by  shaking  the  liquid  with 
amyl  alcohol.  The  orcin  test  gives  an  absorption  band  between 
the  C  and  D  lines,  the  phloroglucin  test,  between  the  D  and  E 
lines.     See  discussion  of  absorption  spectra  below. 

This  procedure  does  not  distinguish  pentoses  from  glucuronic 
acid,  however. 

Arabinose  is  obtained  by  the  hydrolysis  of  gum  arable,  cherry 
gum,  peach  gum,  etc.,  with  dilute  acid.  It  sometimes  occurs  in 
the  urine.  It  has  a  sweet  taste,  and  its  solution  is  dextrorotatory 
(1-arabinose  has  a  specific  rotation  -|-  104.5°).  Its  melting  point 
is  160°.  Its  osazone  melts  at  163°-164°.  Xylose  is  obtained  by 
hydrolyzing  wood,  gum,  straw,  bran,  etc.  It  often  is  called  wood 
sugar.  The  pentose  isolated  from  the  nucleoprotein  of  the  pan- 
creas is  said  to  be  xylose.  Its  solution  is  dextrorotatory.  The 
specific  rotation  of  1-xylose  is  +  18.1°.  Its  phenylosazone  melts 
at  155°-158°  C. 

Absorption  Spectra. 

White  light  is  made  up  of  a  great  many  different  colored 
lights  as  may  be  demonstrated  by  passing  a  beam  of  white  light 
through  a  prism  and  observing  the  spectrum  resulting  from 
spreading  out  the  different  colored  components  of  the  original 
ray.  Colored  light  may  be  either  light  of  a  single  color  or  wave 
length,  or  it  may  be  a  mixture  of  many  different  colored  lights, 
the  sum  of  which,  however,  falls  short  of  making  a  complete 
spectrum,  or  in  which  one  color  greatly  predominates  in  in- 
tensity over  the  other  colors  present.  If  light  passes  through 
water,  it  still  appears  as  white  light,  for  the  water  has  allowed 
the  ray  to  pass  through  intact.  If  a  ray  of  white  light  passes 
through  a  solution  of  hemoglobin,  the  red  pigment  of  the  blood, 
or  of  an  amyl  alcohol  solution  of  the  compound  made  by  heating 
a  pentose  with  orcin  and  hydrochloric  acid,  the  ray  no  longer 
looks  white,  because  the  solution  has  absorbed  or  reflected  a  por- 
tion of  the  colored  light,  allowing  only  the  red  and  perhaps 


CARBOHYDRATES 


51 


some  neighboring  kinds  of  light  to  pass  through.  If  the  light 
is  spread  out  in  a  spectrum,  a  portion  will  be  missing.  Many 
substances  in  solution  have  the  property  of  absorbing  particular 
kinds  of  light  in  this  way,  thus  leaving  a  blank  dark  area  in 
the  spectrum  if  the  emerging  light  is  analyzed  by  spreading  it 
out  into  its  spectrum.  The  property  is  so  constant  that  it  may 
be  made  use  of  to  identify  compounds.  The  spectra  resulting 
are  called  absorption  spectra,  and  each  absorption  spectrum  is 
characteristic  of  a  particular  substance.  The  lines  or  dark 
areas  are  charted  with  reference  to  the  dark  lines  always  ob- 
served in  the  spectrum  of  sunlight.  These  lines  are  called  the 
Frauenhofer  lines.     The  important  ones  are  as  follows: 


C  D  Eb  F 

Red        Yellow        Green        Purple 

By  looking  through  a  solution  with  a  spectroscope  it  thus  is 
possible  to  identify  many  compounds  of  biological  importance. 

Hexoses.  CJI^zOe 

Glucose.  (Dextrose,  grape  sugar). — Glucose  is  found  in  both 
plants  and  animals.  In  the  plant  world  it  occurs  in  grapes  and 
other  sweet  fruits,  in  seeds,  roots,  etc.,  and  as  a  constituent  of 
di-  and  polysaccharides  and  glucosides  is  much  more  widely 
distributed.  In  animals  it  is  found  in  the  blood  and  lymph,  and 
occasionally  in  the  urine.  If  the  amount  in  the  urine  is  more 
than  a  trace  and  it  is  found  regularly,  the  condition  is  pathologi- 
cal and  no  time  should  be  lost  in  consulting  a  physician.  Glu- 
cose also  is  found  in  honey,  an  animal  product.  It  may  be  ob- 
tained by  boiling  starch,  glycogen,  dextrins,  etc.,  with  dilute 
acid.  Glucose  crystallizes  readily.  It  is  soluble  in  water.  The 
solution  is  less  sweet  than  that  of  cane  sugar.  It  is  dextrorota- 
tory, the  specific  rotation  varying  somewhat  with  the  concen- 


52  PHYSIOLOGICAL    CHEMISTRY 

tration.  The  figure  usually  reported  is  -|-52.5°  for  a  solution  in 
which  equilibrium  has  been  reached.  It  shows  strong  mutarota- 
tion.  It  is  slightly  soluble  in  warm  alcohol  and  insoluble  in 
ether.  It  gives  all  the  reduction  tests,  ferments  readily  with 
yeast,  forms  caramel  on  warming  with  alkali,  and  with  phenyl- 
hydrazine  forms  an  osazone  which  melts  at  205°.  It  is  perhaps 
the  most  interesting  of  the  sugars,  for  it  is  found  in  the  blood, 
and  serves  as  one  of  the  most  valuable  fuels  for  the  body  cells. 
By  the  oxidation,  or  burning  of  glucose  the  cells  produce  heat 
and  do  mechanical  work. 

Fructose.  (Levulose,  Fruit  Sugar.) — Fructose  is  found  in 
plants  chiefly  combined  with  glucose  as  cane  sugar,  or  in  the 
polysaccharide  inulin  from  which  it  may  be  obtained  by  hydro- 
lysis. It  also  occurs  in  honey.  It  is  sometimes,  though  rarely, 
found  in  the  urine  in  a  condition  known  as  levulosuria.  The  solu- 
bilities of  fructose  are  similar  to  those  of  glucose.  Its  solution 
rotates  the  plane  of  polarized  .light  to  the  left,  the  specific  rota- 
tion being  — 93°.  It  is  called  d-fructose  because  of  its  struc- 
tural relationship  to  d-glucose,  so  that  in  this  case  the  ''d"  does 
not  indicate  that  the  compound  is  dextrorotatory.  Solutions  of 
fructose  show  the  phenomenon  of  mutarotation.  Fructose  is  a 
ketone  sugar,  and  gives  all  the  usual  reduction  tests  for  car- 
bohydrates. It  forms  the  same  osazone  as  glucose,  so  that  this 
test  is  of  no  value  to  distinguish  the  two  sugars.  Fructose  also 
ferments  with  ordinary  yeast.  Fructose  forms  a  calcium  com- 
pound which  is  much  less  soluble  than  that  of  glucose  and  serves 
to  separate  the  two  sugars  when  they  occur  in  a  mixture. 

Fructose  may  be  distinguished  from  glucose  by  its  levorota- 
tion,  and  also  by  the  Seliwanoff  reaction.  On  adding  a  few 
crystals  of  resorcinol,  and  concentrated  hydrochloric  acid  to  a 
levulose  solution,  and  heating,  a  red  color  results.  Glucose  will 
give  the  test  under  certain  circumstances,  however,  so  that  it 
must  be  carried  out  under  definite  conditions  or  it  may  lead  to 
erroneous  conclusions. 

d-GaJactose. — Galactose  occurs  in  nature  as  a  constituent  of 
several  gums,  in  the  polysaccharide  galactan  in  sea-weed,  as  a 


CARBOHYDRATES  53 

constituent  of  lactose  or  milk  sugar  and  in  certain  substances  in 
brain  and  nerve  tissue.  It  is  prepared  from  milk  sugar  or  from 
various  gums  by  hydrolysis.  Galactose  is  somewhat  less  soluble 
in  water  than  glucose.  The  solution  is  dextrorotatory,  the  spe- 
cific rotation  being  -[-81°-  Galactose  is  an  aldose  and  gives  the 
usual  reduction  tests,  and  forms  with  phcnylhydrazine  an  osa- 
zone  which  melts  at  192°-195°.  Galactose  ferments  slowly  but 
completely  with  ordinary  yeast.  If  heated  with  nitric  acid  it 
forms  mucic  acid  which  is  relatively  insoluble  and  forms  a  fine 
white  precipitate.  This  test  serves  to  distinguish  galactose  from 
all  sugars  except  lactose.  It  is  of  interest  that  the  mammary 
gland  constructs  it  out  of  the  glucose  of  the  blood,  and  unites  it 
with  glucose  to  form  lactose  or  milk  sugar. 

Amino  Sugurs. — Closely  allied  to  the  monosaccharides  are 
the  amino  sugars,  which  differ  from  the  simple  sugars  only  in 
having  an  amino  group  ( — NHj)  instead  of  an  — OH  attached 
to  the  carbon  atom  next  the  aldehyde  group.  These  compounds 
have  been  obtained  by  the  hydrolysis  of  complicated  substances 
occurring  in  the  shells  of  lobsters  and  from  the  proteins  mucin 
and  mucoid  which  are  widely  distributed  in  the  animal  world. 
d-Glucosamine  is  an  important  member  of  this  group.  It  is  ob- 
tained by  boiling  the  chitin  of  lobster  shells  with  hydrochloric 
acid.  It  is  readily  soluble  in  water,  the  solution  being  alkaline. 
Its  hydrochloric  acid  salt  shows  solubilities  similar  to  those  of 
the  monosaccharides.  The  solution  is  dextrorotatory,  having  a 
specific  rotation  of  from  70° -\-  to  74° -|-  according  to  concen- 
tration. It  reduces  the  ordinary  carbohydrate  reagents  and  gives 
an  osazone  identical  with  that  formed  from  glucose.  Gluco- 
samine does  not  ferment,  however.  d-Glucosamine  is  an  inter- 
esting compound  because  in  composition  it  stands  midway  be- 
tween the  carbohydrates  and  a  group  of  substances  called  amino 
acids,  which  are  the  simple  units  of  which  the  proteins  are  com- 
posed. 

d-Glucuronic  Acid. — This  compound  is  obtained  from  glucose 
by  oxidation,  and  is  found  in  the  body  in  combination  with 
other  substances.  These  compounds  are  called  conjugated  glu- 
curonates.     Glucuronic  acid  has  the  formula: 


54  PHYSIOLOGICAL    CHEMISTET 

COOH 

I 
H— C— OH 

I 
H— C— OH 

HO— C— H 

I 
H_C— OH 

I 
CHO 

Glucuronic  acid 

It  is  interesting  chiefly  because  it  combines  with  various  pro- 
duets  formed  by  putrefaction  in  the  intestine  such  as  phenol, 
indol,  skatol,  etc.  These  substances  are  absorbed  into  the  blood 
stream,  and  would  exert  a  toxic  influence  upon  the  cells  if  it 
were  not  for  the  fact  that  the  body  promptly  unites  them  to 
glucuronic  acid,  (or  some  other  compounds)  forming  the  con- 
jugated glucuronates  which  are  relatively  harmless,  and  are 
excreted  in  the  urine.  This  is  one  of  nature's  protective  de- 
vices for  shielding  the  cells  against  the  influence  of  injurious 
substances.  Solutions  of  glucuronic  acid  give  the  same  reduc- 
tion tests  as  glucose,  are  dextrorotatory,  but  do  not  ferment. 
The  conjugated  glucuronates  are  strongly  levorotatory. 

Disaccharides. 

The  disaccharides  are  formed  by  the  union  of  two  molecules 
of  monosaccharide  with  loss  of  water. 

CeHiaOg  +  CgHiaOe  -^  C12H22O11  -\-    HgO. 

By  the  action  of  dilute  acids,  enzymes,  etc.,  they  may  be 
split  into  their  constituent  monosaccharides.  The  three  disac- 
charides of  importance  are  saccharose,  lactose  and  maltose. 
These  sugars  are  of  great  importance  as  food  substances. 

Saccharose.  (Sucrose,  Cane  Sugar.) — Cane  sugar  is  found 
in  many  plants,  notably  in  the  juice  of  the  sugar  cane,  which 
contains  about  20%,  and  in  carrots.  It  is  found  in  many  sweet 
fruits,  such  as  the  banana,  strawberry,  pineapple,  etc.,  and  in 


CARBOHYDRATES 


55 


the  sap  of  the  sugar  maple.  It  is  a  valuable  food  substance, 
and  serves  also  as  a  condiment,  by  its  sweetness  making  other 
foods  more  palatable.  Cane  sugar  is  prepared  by  treating  the 
sap  or  juice  containing  it  with  milk  of  lime.  This  neutralizes 
any  acids  present,  which  otherwise  would  hydrolyze  the  sugar 
during  evaporation.  After  being  boiled  to  remove  the  protein, 
the  calcium  is  removed  by  running  in  carbon  dioxide,  and  the 
solution  is  decolorized  either  with  animal  charcoal  or  sulphur 
dioxide.  After  being  boiled  and  filtered  the  liquid  is  evaporated 
in  vacuo,  and  the  cane  sugar  crystallizes  out.  The  remaining 
liquid  is  known  as  molasses,  and  still  contains  considerable  quan- 
tities of  sugar  which  may  be  obtained  by  precipitation  as  cal- 
cium or  strontium  saccharate.  From  this  compound  the  cane 
sugar  may  be  set  free  with  carbon  dioxide. 

Cane  sugar  is  readily  soluble  in  water,  less  so  in  alcohol,  and 
insoluble  in  ether.  The  aqueous  solution  is  very  sweet,  and 
is  strongly  dextrorotatory,  the  specific  rotation  being  -j-  66.5°. 
The  specific  rotation  of  saccharose  is  practically  independent  of 
changes  in  concentration  and  temperature,  so  that  the  property 
is  often  made  use  of  for  its  estimation.  On  hydrolysis  it  yields 
glucose  and  fructose. 

On  being  heated  to  about  160°  cane  sugar  melts  and  if  al- 
lowed to  cool,  forms  a  glassy  mass  which  is  known  as  barley 
sugar.    At  about  200°  it  turns  brown,  forming  caramel. 


56  PHYSIOLOGICAL    CHEMISTRY 

Cane  sugar  does  not  reduce  the  usual  carbohydrate  reagents, 
such  as  Fehling's  or  Nylander's  solutions  and  it  does  not  form 
an  osazone  if  treated  with  phenylhydrazine.  This  is  due  to  its 
molecular  structure. 

The  aldehyde  and  ketone  groups  of  its  constituents  are  no 
longer  free.  On  long  boiling  with  these  reagents,  however,  or. 
after  the  action  of  an  inverting  enzyme  on  the  cane  sugar,  the 
solution  will  give  positive  reactions  with  the  above  reagents 
since  the  sugar  is  split  into  its  constituent  parts.  The  hydrol- 
ysis or  splitting  of  cane  sugar  is  called  inversion  because  the 
solution,  originally  dextrorotatory,  is  levorotatory  after  hydroly- 
sis. This  is  due,  of  course,  to  the  fact  that  the  hydrolyzed  solution 
contains  equal  quantities  of  glucose  and  fructose,  the  latter 
of  which  rotates  more  strongly  to  the  left  than  does  glucose  to 
the  right.  The  mixture  is  known  as  "invert  sugar."  A  solu- 
tion of  cane  sugar  ferments  readily  with  ordinary  yeast,  which 
contains  an  enzyme  invertase  which  will  invert  the  cane  sugar. 
The  resulting  glucose  and  fructose  are  fermented  by  the  zymase. 

Lactose. — Lactose  is  found  in  the  milk  of  all  mammals,  but 
does  not  occur  in  plants.  Cow's  milk  contains  about  4%  lac- 
tose, human  milk  about  5%  to  7%.  It  may  occur  in  the  urine  of 
women  during  pregnancy.  Lactose  is  prepared  from  whey. 
On  concentration,  lactose  crystallizes  out,  and  may  be  purified 


H 

CH20H 

C  =  0 

1 

CHOH 

1 

CHOH 

1 

CH 

CHOH 

1 

CHOH           ( 

D                   CHOH 

CHOH 

CHOH 

1 

CH— 0 

CH„ 

Galactose 

Glucose 

Lactose 


CARBOHYDRATES  57 

by  recrystallization  from  water.  The  crystals  are  hard  and 
gritty,  and  the  solution  is  not  so  sweet  as  that  of  cane  sugar. 
Lactose  is  composed  of  one  molecule  each  of  glucose  and  galac- 
tose. It  is  manufactured  in  the  breast  gland  from  the  glucose 
of  the  blood.  This  is  an  interesting  example  of  the  conversion 
of  one  sugar  into  another  in  the  body,  since  a  portion  of  the 
glucose  must  bo  changed  into  galactose. 

Lactose  responds  readily  to  the  reduction  tests  for  the  mono- 
saccharides. It  thus  possesses  a  free  aldehyde  group,  and  is 
represented  by  the  accompanying  formula.  The  solution  of 
lactose  is  dextrorotatory.  The  specific  rotation  is  -|-  52.5°.  Lac- 
tose does  not  ferment  with  ordinary  yeast.  This  property  is 
serviceable  in  identifying  it.  It  should  be  remembered  that  lac- 
tose will  give  the  mucic  acid  test,  since  it  contains  galactose. 

Maltose. — Maltose  is  formed  in  the  hydrolysis  of  starch  or 
glycogen  by  amylase.  Since  this  enzyme  is  widely  distributed 
in  both  plants  and  animals,  maltose  may  be  found  wherever 
there  is  starch  or  glycogen. 

Maltose  is  readily  soluble  in  water.  The  solution  is  not  so 
sweet  as  that  of  cane  sugar.  The  solution  is  dextrorotatory,  the 
specific  rotation  being  -|-137°.  This  value  varies,  however,  with 
concentration  and  temperature.  Maltose  reduces  Fehling's  re- 
agent, etc.,  and  gives  an  osazone.  Its  structure  is  thus  con- 
sidered to  be  similar  to  that  of  lactose  which  has  one  free  al- 
dehyde group.  On  hydrolysis  it  yields  two  molecules  of  glu- 
cose. Maltose  ferments  readily  with  yeast.  It  may  easily  be  dis- 
tinguished from  glucose  by  hydrolizing  with  dilute  acid.  After 
hydrolysis  the  reducing  power  of  the  solution  will  be  found  to 
have  increased,  since  two  molecules  of  glucose  are  now  present 
for  every  molecule  of  maltose  destroyed. 

Polysaccharides. 

Members  of  the  polysaccharide  group  differ  from  one  another 
considerably  in  their  solubilities  and  other  properties.  They 
are  found  in  both  plants  and  animals  in  which  they  form  reserve 
supplies  of  food  material,  and  in  plants  and  some  of  the  lower 


58  PHYSIOLOGICAL    CHEMISTRY 

animals,  they  are  important  constituents  of  the  supporting 
framework  or  protective  covering.  The  members  of  this  group 
are  made  up  of  several  molecules  of  monosaccharide  united  with 
loss  of  water  to  form  the  larger  polysaccharide  molecule.  The 
commoner  individual  polysaccharides  yield  only  one  kind  of 
monosaccharide  when  hydrolized,  thus  differing  from  the  other 
material  bases  which,  on  hydrolysis  yield  varying  kinds  of  sim- 
pler units.  Since  the  number  of  monosaccharide  molecules  which 
make  up  a  polysaccharide  molecule  is  unknown,  it  is  customary 
to  express  the  formula  with  the  indefinite  coefficient  n. 

Starch  (06Hio05)n. — Starch  is  a  plant  product,  and  is  found 
stored  in  leaves,  seeds,  fruits,  tubers,  etc.  Grains  contain  as 
much  as  50-70%  of  their  dry  weight;  potatoes  contain  from 
15-30%  of  their  wet  weight.  Starch  forms  granules  of  more  or 
less  characteristic  shapes  which  are  serviceable  in  determining 
the  source  of  the  starch.  Thus  potato  starch  appears  as  egg- 
shaped  granules  which  often  show  concentric  lines.  Starch  is 
prepared  from  potatoes  or  grain  by  grinding  the  material,  filter- 
ing through  sieves  to  remove  the  coarse  debris  and  allowing  the 
suspended  starch  particles  to  settle.  It  forms  a  white  amorphous 
powder  which  does  not  dissolve  in  cold  water.  If  boiled  with 
water,  the  granules  are  broken  open  and  the  starch  forms  an 
opalescent  solution.  Starch  is  insoluble  in  alcohol  and  ether. 
A  starch  solution  rotates  the  plane  of  polarized  light  to  the 
right.  It  will  not  reduce  Fehling's,  or  similar  solutions,  and 
will  not  ferment.  The  characteristic  starch  test  is  the  formation 
of  a  blue  color  on  the  addition  of  a  few  drops  of  iodine  solu- 
tion. This  color  disappears  on  heating,  but  reappears  if  the 
solution  is  cooled.  Alkali  and  alcohol  also  destroy  the  blue 
color.  On  boiling  with  dilute  acids  starch  is  broken  down  to 
glucose.  The  starch  passes  through  various  intermediate  stages, 
the  nature  of  which  may  be  foUow^ed  by  the  iodine  test.  If 
portions  of  the  hydrolizing  mixture  are  tested  with  iodine  from 
time  to  time,  the  blue  color  soon  gives  place  to  red.  This  cor- 
responds to  the  stage  known  as  erythrodextrin  (from  the  Greek 
word  meaning  ''red").    On  further  hydrolysis,  the  iodine  test 


CARBOHYDRATES  59 

gives  no  color.  This  is  the  achroodextrin  stage  (Greek  word 
means  "no  color").  Beyond  this  stage  maltose  is  formed, 
which  then  breaks  up  into  glucose.  The  appearance  of  reducing 
sugars  may  be  recognized,  since  the  mixture  will  reduce  Feh- 
ling's  solution. 

Starch  is  an  enormously  important  food  substance,  and  is 
widely  used  in  the  arts.  In  stiffening  linen,  the  starch  is  broken 
down  into  dextrins  by  the  heat  of  the  iron.  These  dextrins 
give  the  fabric  its  stiffness  and  glossy  appearance. 

Dextrins.— Little  need  be  said  of  the  dextrins  in  addition  to 
the  fact  that  they  are  intermediate  stages  formed  in  the  hydrol- 
ysis of  starch  to  glucose.  There  probably  are  many  members 
of  the  group,  but  so  far,  little  is  known  of  the  different  individ- 
ual dextrins.  Dextrins  themselves  probably  do  not  reduce  Feh- 
ling's  solution,  or  only  slightly,  but  commercial  dextrin,  which 
is  prepared  by  the  partial  hydrolysis  of  starch,  usually  reduces 
Fehling's  solution  slightly,  probably  because  the  mixture  con- 
tains maltose  or  glucose  as  the  result  of  complete  hydrolysis  of 
some  of  the  material.  Dextrin  solutions  do  not  ferment,  and 
give  a  red  color  (erythrodextrin)  or  no  color  (achroodextrin) 
with  iodine  according  to  the  extent  of  the  hydrolysis.  Dex- 
trins are  readily  soluble  in  water,  but  are  precipitated  by  the 
addition  of  alcohol.     The  aqueous  solution  is  dextrorotatory. 

Inulin.^ — Inulin  occurs  in  the  sap  of  various  plants  and  is 
found  to  the  extent  of  10-12%  in  the  tubers  of  the  dahlia.  It 
is  soluble  in  hot  water,  and  gives  a  yellow  or  brownish  color 
with  iodine.  It  is  interesting  chiefly  because  on  hydrolysis  it 
yields  levulose  instead  of  glucose.  It  does  not  reduce  Fehling's 
solution,  and  its  solution  is  levorotatory. 

Gums  and  Mucilages. — The  gums  and  mucilages  are  widely 
distributed  and  on  hydrolysis  yield  various  pentoses  and  hex- 
oses,  and  other  substances.  They  vary  greatly  in  their  solubil- 
ities. 

Cellulose. — Cellulose  is  chiefly  important  in  forming  a  large 
part  of  the  structural  framework  of  plants.  The  plant  cell  walls 
are  made  up   of  cellulose  mixed  with  lignin  and  other  sub- 


60  PHYSIOLOGICAL   CHEMISTEY 

stances.  Cellulose  also  is  found  in  certain  lower  animals,  the 
tunicates.  Cellulose  is  insoluble  in  the  ordinary  solvents.  It 
dissolves,  however,  in  Schweizer's  reagent,  an  ammoniacal  solu- 
tion of  copper  oxide,  and  in  some  other  reagents.  Cellulose 
derivatives  are  extensively  used  in  the  arts.  Thus  nitrocellu- 
loses  of  varying  composition  are  used  in  the  manufacture  of  ex- 
plosives, collodion,  celluloid,  artificial  rubber,  etc.  From  cellu- 
lose artificial  silk  and  artificial  gutta  percha  also  are  prepared. 

There  has  been  much  discussion  as  to  whether  cellulose  is  of 
value  as  a  food.  Undoubtedly  this  is  the  case  in  herbivora.  In 
man  it  probably  is  of  little  food  value,  but  serves  a  useful  pur- 
pose in  giving  bulk  to  the  food  and  thus  stimulating  the  mus- 
cular activity  of  the  intestine.  It  has  been  stated  that  the  cellu- 
lose of  young  and  tender  lettuce,  asparagus,  etc.,  may  be  utilized 
by  the  body  to  a  considerable  extent  as  food.  There  is  no  enzyme 
in  the  digestive  juices  capable  of  hydrolyzing  cellulose,  so  that 
its  disintegration  must  be  due  to  the  action  of  intestinal  bac- 
teria. 

Glycogen. — Glycogen  is  found  in  the  organs  and  tissues  of 
animals,  and  also  in  some  plants  (yeast).  It  serves  as  a  reserve 
fuel  or  food  supply.  The  chief  depots  for  glycogen  deposit 
are  the  liver  and  the  muscles.  Glycogen  is  found  in  oysters, 
scallops  and  other  molluscs.  Glycogen  may  be  prepared  from 
the  liver  of  an  animal  which  has  just  been  killed.  The  liver  is 
ground  in  a  mortar  with  sand,  and  extracted  with  boiling  water 
slightly  acidified  with  acetic  acid.  If  the  extraction  is  not  made 
at  once  after  the  death  of  the  animal  the  glycogen  supply  will 
be  greatly  diminished  or  disappear  altogether,  as  it  is  rapidly 
hydrolized  to  glucose  by  enzymes  in  the  liver  tissue.  Feeding 
rabbits  for  a  day  or  two  on  carrots  before  killing  will  insure  a 
liberal  supply  of  glycogen  in  the  liver. 

Glycogen  is  a  white  amorphous  powder,  which  dissolves  in 
cold  water  forming  an  opalescent  solution.  This  solution  gives 
a  wine  red  or  brown  color  with  iodine.  It  does  not  reduce  Feh- 
ling  's  solution,  is  not  fermented  by  yeast,  and  is  dextrorotatory. 
On  boiling  with  dilute  acids,  glycogen  is  hydrolyzed  to  glucose. 


CARBOHYDRATES  61 

After  hydrolysis  the  solution  will  of  course,  reduce  Fehling's 
solution. 

Glucosides. 

Many  compounds  are  obtained  from  plants  and  animals  which 
yield  on  hydrolysis  varying  quantities  of  glucose  or  other  sim- 
ple sugars  and  in  addition  a  wide  variety  of  other  compounds. 
To  this  group  is  given  the  name  glucosides.  Among  these  sub- 
stances are  many  of  the  drugs  used  in  medicine.  The  di-  and 
polysaccharides  themselves  may  be  looked  upon  as  glucosides, 
since  in  them  glucose  is  combined  with  one  or  more  sugar 
molecules.  ,  ! 


CHAPTER  IV 
FATS,  PHOSPHATIDS  AND  ALLIED  SUBSTANCES 

Distribution  and  Importance. — The  fats  are  widely  distrib- 
uted in  nature,  in  both  plants  and  animals.  In  the  former  they 
are  found  in  seeds  such  as  cotton  seed,  the  castor  bean,  etc.,  in 
fruits,  such  as  olives,  in  nuts  and  also  in  the  leaves  and  roots  of 
some  plants.  In  animals  they  are  found  in  most  tissues  and 
fluids.  The  amounts  in  the  tissues  vary  considerably.  The  ac- 
tive living  protoplasm  contains  only  about  1-10%,  w^hereas  mar- 
row, fatty  tissue,  etc.,  may  contain  considerably  over  90%.  The 
fats  are  of  importance  as  fuels  for  the  body.  They  are  laid 
away  in  large  deposits  which  also  serve  the  purpose  of  insulat- 
ing the  body  by  forming  a  blanket  layer  which  aids  in  the  con- 
servation of  heat.  There  is  a  layer  of  subcutaneous  fat,  and 
there  are  also  large  deposits  around  the  abdominal  viscera. 
Considerable  quantities  are  found  in  the  intramuscular  con- 
nective tissue. 

Composition  and  Structure. — The  fats  are  made  up  of  car- 
bon, hydrogen  and  oxygen.  The  oxygen  is  present  in  much 
smaller  per  cent  than  in  the  carbohydrates.  The  constituent 
parts  of  the  fats  are  the  triatomic  alcohol  glycerine  or  occa- 
sionally some  other  alcohol,  and  organic  acids,  either  of  the  fatty 
acid  or  a  similar  series.  It  is  of  interest  that  the  acids  making 
up  the  body  fats  have  even  numbers  of  carbon  atoms  in  their 
molecules.  The  following  list  gives  the  names  and  formulas  of 
some  of  the  important  acids: 

Butyric  OH.CH^CH.COOH  (C.HgOJ 

Caproic  CHsCH^CHjCHgCH^COOH        (CJl.^O,) 

Caprylic  CHgCCHJeCOOH  (CgHigOs) 

Capric  CH3(CH2)8C00H  (CH^oO,) 

Palmitic  CH3(CH2)i4C00H  (Ci.Hg^OJ 

Stearic  CH3(CH2),eC00H  (CigHaeOJ 

62 


FATS,    PHOSPHATIDS,    AND   ALLIED   SUBSTANCES  63 

The  acids  listed  above  are  all  saturated  compounds,  that  is  they 
contain  no  double  bonds.  An  acid  found  in  a  large  number  of 
fats  is  oleic  acid,  which  has  the  same  number  of  carbon  atoms 
as  stearic  acid,  but  two  less  hydrogen  atoms.  It  is  thus  CigHo^Oa 
and  its  formula  is 

CHsCCHJ.CH  =  CHCCHJ.COOH. 

It  is  an  unsaturated  acid,  and  contains  a  double  bond.  Fats 
containing  this  acid  have  a  lower  melting  point  than  those  con- 
taining the  corresponding  saturated  compound.  Some  allied 
compounds  contain  other  alcohols  in  place  of  glycerine.  Thus 
cetyl  alcohol  Cj^JI^.fiJi  is  found  in  spermaceti  in  the  head  of 
the  sperm  whale,  and  myricil  alcohol  CgoHgiOH  in  beeswax,  etc. 
Esters  of  these  alcohols  usually  are  called  waxes.  The  follow- 
ing formula  illustrates  the  structure  of  a  fat. 

0 

II 
CH2O— C— R 
O 

II 
CH— 0— C— R 
I  0 

I  II 

CH2— 0— C— R 

R  is  the  rest  of  an  acid  molecule.    If  the  fat  were  tristearin,  R 

would  represent  a  chain  of  16  CHg  groups  with  a  CH3  group  at 

the  far  end.    Fats  are  thus  tri-atomic  esters  of  glycerine  and  an 

organic  acid.    The  three  R's  may  be  all  the  same  fatty  acid,  or 

they  may  be  different.    There  is  thus  the  possibility  of  having  a 

large  number  of  different  fats,  differing  in  the  kind  of  fatty 

acid  present.     This  possibility  is  realized  in  nature,  and  a  large 

number  of  different  fats  are  known.     The  naturally  occurring 

fats  are  rarely  made  up  of  a  single  kind  of  fat,  but  usually  are 

mixtures  of  various  kinds  such  as  tripalmitin,  tristearin,  and 

triolein,  as  the  fats  from  these  respective  acids  are  called.    Oleic 

acid  has  a  very  low  melting  point,  and  triolein  also  melts  at  a 

low  temperature.    The  presence  of  much  triolein  in  a  fat  lowers 


64  PHYSIOLOGICAL    CHEMISTRY 

its  melting  point,  often  to  such  an  extent  that  the  fat  is  liquid 
at  ordinary  room  temperature.  Such  fats  are  called  oils. 
Other  unsaturated  acids  are  found  in  some  fats,  or  "oils,"  and 
they  exert  a  similar  influence. 

General  Properties. — The  solid  fats  are  white  or  light  yellow 
substances,  which  if  pure  are  odorless  and  tasteless.  The  oils 
often  are  yellow  and  frequently  have  a  decided  taste  and  odor. 
They  are  insoluble  in  water,  somewhat  soluble  in  cold  alcohol 
but  much  more  so  in  hot  alcohol,  and  soluble  in  ether,  chloro- 
form, benzol,  etc.  From  solutions,  the  fats  often  may  be  ob- 
tained in  crj^stalline  form  as  long  needles.  The  specific  grav- 
ities of  all  the  fats  and  oils  are  less  than  that  of  water,  hence 
they  float  at  the  surface.  They  reduce  the  surface  tension  of 
water.  The  naturally  occurring  fats  and  oils  do  not  have  sharp 
melting  points,  since  they  are  mixtures  of  different  kinds  of 
fats.  Even  pure  fats  often  show  much  indefiniteness  in  melting 
point.  Some  melt,  resolidify  at  a  slightly  higher  temperature, 
and  if  further  warmed,  melt  again.  This  is  supposed  to  be  due 
to  the  fact  that  an  internal  rearrangement  in  the  molecule  is 
brought  about  by  heating. 

Emulsification. — If  neutral  oil  and  water  are  shaken  to- 
gether vigorously,  and  the  mixture  allowed  to  stand,  it  will 
quickly  separate  into  two  layers,  oil  and  water.  If  a  small 
amount  of  soap  solution  is  added  to  this  mixture  and  the  shak- 
ing repeated,  the  liquid  becomes  milky  in  appearance,  and  even 
after  prolonged  standing  will  fail  to  separate  into  two  layers. 
The  fat  is  said  to  be  emulsified.  On  examining  such  a  mix- 
ture under  the  microscope,  it  will  be  seen  to  be  filled  with 
minute  globules  of  oil  suspended  in  the  water.  If  the  propor- 
tions are  reversed,  that  is,  if  there  is  much  oil  and  little  water, 
the  water  will  be  suspended  in  the  oil.  Soap  is  by  no  means 
the  only  substance  which  will  favor  the  formation  of  an  emul- 
sion. Albumin,  gums  such  as  gum  arabic,  and  a  variety  of 
other  compounds  will  bring  about  a  similar  result.  Lymph 
will  emulsify  a  fat,  and  if  a  drop  of  lymph  and  a  drop  of  oil 
are  brought  in  contact  on  a  microscope  slide,  the  oil  may  be  seen 


FATS,    PHOSPHATIDS,    AND   ALLIED    SUBSTANCES  65 

to  break  up  into  minute  droplets  and  enter  the  l^^mph  drop. 

Physiologically  the  formation  of  emulsions  is  of  great  im- 
portance. In  digestion  in  the  stomach  only  emulsified  fats  are 
attacked  to  any  extent.  In  the  intestine,  fats  of  the  food  are 
emulsified  by  the  pancreatic  juice,  a  process  which  is  extremely 
important  for  their  proper  digestion  and  absorption.  The 
mechanism  of  emulsion  formation  has  been  the  subject  of  much 
study.  Probably  different  emulsifying  agents  act  in  different 
ways,  or  a  single  substance  may  act  in  more  than  one  way. 
It  is  believed  that  the  soap,  albumin,  etc.,  collects  around  the 
tiny  fat  droplets  and  serves  to  insulate  them  and  thus  lessen 
the  tendency  to  run  together.  The  lowering  of  the  surface 
tension  is  also  a  factor  in  the  production  of  emulsions,  and 
possibly  also  electrical  forces  tending  to  repel  the  similarly 
charged  particles  of  fat.  Milk  is  an  example  of  a  fairly  perma- 
nent emulsion.  The  fat  droplets  are  suspended  in  a  liquid 
which  contains  protein.  On  standing,  a  considerable  portion 
of  the  milk  fat  finally  Mall  float  to  the  surface.  When  removed 
from  the  skimmed  milk  beneath,  this  is  known  as  cream.  On 
churning,  the  emulsified  fat  runs  together  and  butter  is  formed. 

Saponification. — If  a  fat  is  boiled  with  an  alkali,  or  an  acid, 
it  is  split  into  fatty  acids  and  glycerine.  This  process  is  known 
as  saponification.  If  an  alkali  is  used,  the  fatty  acids  react 
with  the  alkali  to  form  salts.  These  salts  of  the  higher  fatty 
acids  have  a  slippery  feeling,  and  their  solutions  foam  on  being 
shaken.  They  are  called  soaps.  The  accompanying  equation 
illustrates  the  process: 


0 

II 

CH^O  —  C.  C,,H.,  CH.OH 

0  I 

CHO  —  C  .  C,-Il3,  3  NaOH  -^  CHOH  +  SCVHg-COONa 

I  0  =  I 

I  I!  I 

CH^O  —  C  .  Ci,H3,  CH^OH 


66  PHYSIOLOGICAL    CHEMISTRY 

The  process  is  best  carried  out  in  alcoholic  solution.  Sodium 
soaps  are  known  as  hard  soaps;  potassium  soaps  which  are  but- 
tery in  consistency  are  known  as  soft  soaps.  Calcium  soaps 
are  very  hard  and  insoluble.  Soap  is  a  useful  cleansing  agent. 
Soiled  articles, — clothing,  the  hands,  etc.,  usually  are  covered 
with  a  layer  of  fatty  material  which  entangles  and  holds  parti- 
cles of  insoluble  inorganic  dirt.  Soap  emulsifies  the  fat  and 
the  remaining  material  is  carried  away  by  the  water  or  by  the 
lather,  which  takes  up  the  particles  of  dirt  mechanically. 

Since  calcium  soaps  are  very  insoluble,  hard  water  is  not 
good  for  washing  purposes,  as  the  calcium  precipitates  the  soap 
added,  and  thus  interferes  with  its  cleansing  activities. 

Rancid  Fats. — Many  natural  fats,  upon  standing,  acquire  a 
disagreeable  taste  and  odor.  This  is  due  to  the  splitting  of 
some  of  the  neutral  fat  into  glycerine  and  fatty  acids.  The 
lower  fatty  acids,  such  as  those  found  in  butter  have  a  very 
disagreeable  taste  and  odor,  hence  the  character  of  ''rancid" 
butter,  etc. 

Detection  and  Identification. — Acrolein  Test. — Fats  are  easily 
detected  by  their  physical  properties,  such  as  solubility,  appear- 
ance, greasiness,  etc.  A  test  given  by  all  common  fats  is 
known  as  the  acrolein  test.  If  a  fat  is  heated  to  300°  it  is  de- 
composed. The  glycerine  portion  of  the  molecule  loses  water 
and  forms  the  unsaturated  compound  acrolein.  The  test  is 
obtained  more  readily  if  the  fat  is  heated  with  a  dehydrating 
agent  such  as  potassium  acid  sulphate,  boric  acid  or  phosphorus 
pentachloride.  Acrolein  is  easily  recognized  by  its  extremely 
sharp  and  irritating  odor.  Since  only  substances  containing  glyc- 
erine give  the  test,  it  may  be  used  to  distinguish  between  fats 
and  fatty  acids  or  soaps. 

CH.OH  CH2 

I     "  II 

CHOH  _2H,0^  CH 

I  "  I 

CH2OH  CHO 

Acrolein. 


FATS,    PHOSPHATIDS,    AND   ALLIED    SUBSTANCES  67 

Melimg  Point. — The  melting  points  of  the  natural  fats  are 
not  sharp,  since  natural  fats  usually  are  mixtures.  They  often 
melt,  solidify  on  further  heating,  and  melt  again  at  a  higher 
temperature.  Those  fats  whose  melting  points  are  below  or- 
dinary room  temperature  are  called  oils.  The  melting  points 
of  fats  in  animal  tissues  are  generally  below  the  usual  tempera- 
ture of  those  tissues,  so  that  the  body  fats  are  in  a  fluid  state. 
The  fats  of  cold  blooded  animals  melt  at  low^er  temperatures 
than  those  of  warm  blooded  animals. 

Saponification  Equivalent. — The  saponification  equivalent  is 
the  number  of  milligrams  of  potassium  hydrate  necessary  to 
neutralize  the  fatty  acids  produced  by  the  saponification  of  one 
gram  of  fat.  The  smaller  the  molecular  weight  of  the  acids 
in  the  fat,  the  larger  will  be  the  number  of  molecules  in  a  gram, 
and  the  higher  the  saponification  number.  Fats  made  up  of 
fatty  acids  such  as  palmitic,  stearic  and  oleic  acid  such  as  oleo- 
margarine have  a  saponification  number  around  195.  Butter, 
which  contains  fatty  acids  of  low  molecular  weight  has  a 
saponification  number  around  227.  These  two  substances  may 
be  distinguished  easily  in  this  w^ay. 

Volatile  Fatty  Acids. — Reichert-Meissl  Nuniber. — This  method 
is  used  to  give  evidence  of  the  amount  of  low^er  fatty  acids  in 
a  fat.  The  fat  is  hydrolized  with  alkali,  acidified  with  sul- 
phuric acid  and  distilled.  The  fatty  acids  of  low  molecular 
weight  distil  over  and  may  be  titrated.  Those  of  higher  molec- 
ular weight  are  not  volatile  and  remain  behind.  The  number 
of  cubic  centimeters  of  N/10  alkali  required  to  neutralize  the 
volatile  fatty  acids  from  5  grams  of  fat  is  called  the  Reichert- 
Meissl  number. 

Iodine  Numher. — The  acids  which  contain  double  bonds,  such 
as  oleic  acid  will  take  up  iodine  or  bromine,  adding  on  two 
atoms  of  the  halogen  for  each  double  bond.  By  this  process  it 
is  possible  to  determine  how  much  unsaturated  acid  is  present 
in  a  fat.  The  weight  of  iodine  in  centigrams  taken  up  by  a 
gram  of  oil  is  known  as  the  iodine  number.     Hvdrogen  and 


68  PHYSIOLOGICAL   CHEMISTRY 

oxygen  are  taken  up  in  an  analogous  manner,  and  this  fact  is 
also  used  to  identify  fats. 

Acetyl  Eqivalent. — Some  fats  contain  oxyacids, — that  is 
acids  containing  hydroxyl  groups.  These  groups  may  be  re- 
placed by  acetyl  groups,  which  in  turn  may  be  split  off  and 
the  resulting  acetic  acid  titrated.  The  number  of  milligrams 
of  potassium  hydrate  required  to  neutralize  the  acetic  acid  ob- 
tained from  1  gram  of  fat  in  which  the  hydroxyl  groups  have 
been  replaced  by  acetyl  groups  is  known  as  the  acetyl  equiva- 
lent. 

The  various  factors  described  above  have  been  determined 
for  the  important  natural  fats,  and  variations  in  one  or  more 
of  these  characteristic  constants  are  of  great  service  in  deter- 
mining whether  a  given  fat  or  oil  is  pure,  or  has  been  adul- 
terated. 

Important  Fats. — Tristearin,  tripalmitin  and  triolein  are  the 
three  fats  occurring  most  frequently  in  natural  fats. 

Tristearin,  or  stearin,  as  it  is  often  called,  melts  first  at 
about  55°,  resolidifies  and  melts  again  at  about  71°.  It  is  a 
hard,  flaky  material,  and  the  least  soluble  of  the  three.  It  is 
obtained  from  tallow.  Mixed  with  a  little  paraffin  to  make  it 
less  brittle  it  is  moulded  into  candles.  Free  stearic  acid  is  found 
in  old  pus,  in  gangrenous  or  tuberculous  masses,  etc.,  where 
decomposition  of  fat  has  taken  place.  It  is  found  as  its  alkali 
soap  in  blood,  bile,  etc.,  and  as  its  calcium  soap  in  the  feces. 

Tripalmitin,  or  palmitin,  is  found  in  all  animal  and  most 
vegetable  fats,  notably  in  palm  oil,  whence  it  derives  its  name. 
It  predominates  in  human  fat.  It  melts  first  at  about  50°,  re- 
solidifies, and  melts  again  at  about  66°. 

Triolein,  or  olein,  is  found  in  animal,  and  to  a  greater  extent 
in  vegetable  fats  and  oils.  It  melts  at  — 6°  C,  and  is  thus  a 
liquid  (an  oil)  at  room  temperature.  The  presence  of  olein 
lowers  the  melting  point  of  natural  fats.  If  exposed  to  the  air 
olein  quickly  becomes  rancid.  Oleic  acid,  by  reason  of  its 
double  bond  will  take  up  iodine  as  described  above. 

Butter. — Butter,  the  fat  obtained  from  cream,  contains  about 


FATS,   PHOSPHATIDS,    AND   ALLIED   SUBSTANCES  69 

80%  fats.  About  7%  of  this  is  made  of  lower  fatty  acids.  Of 
the  remainder  60-70%  is  palmitin,  and  30-40%  olein.  Butter 
contains  very  little  stearin. 

Oleomargarine  is  made  usually  from  beef  fat.  The  fat  is 
melted,  cooled,  the  oily  portion  pressed  out  and  this  mixed  with 
various  substances  such  as  peanut  oil,  lard,  etc.,  and  finally 
churned  up  with  milk  to  give  it  a  butter  flavor.  If  oleomarga- 
rine is  made  from  clean,  wholesome  materials  it  is  a  perfectly  sat- 
isfactory food  substance.  It  lacks  certain  substances  found  in 
butter,  however,  which  are  called  vitamines,  which  recently 
have  been  shown  to  be  very  valuable  materials  to  the  body. 
Cod  liver  oil  also  contains  these  substances.  Perhaps  this  fact 
justifies  the  popular  impression  of  its  beneficial  nature. 

Lanolin,  or  wool  fat,  which  is  an  ester  of  higher  fatty  acids 
chiefly  with  the  alcohol  cholesterol  in  place  of  glycerine,  is  use- 
ful in  medicine.  It  forms  extremely  fine  emulsions  and  thus 
serves  as  a  valuable  basis  for  the  formation  of  salves  and  oint- 
ments. Substances  containing  alcohols  other  than  glycerine, 
such  as  cholesterol  are  often  classed  as  waxes. 

Lecinthin  and  Cholesterol 

A  large  number  of  substances  have  been  isolated  from 
animal  and  plant  tissues  which  have  certain  similarities  to 
the  fats,  such  as  solubilities,  general  appearance,  etc.  Some 
of  these  compounds  are  also  related  chemically  to  the 
fats.  Substances  of  this  nature  are  found  in  all  cells,  both 
animal  and  vegetable,  and  particularly  in  the  brain  and  nerve 
tissues.  Little  is  known  of  the  biological  function  of  these  com- 
pounds, although  as  our  information  increases,  they  appear  to 
be  increasingly  important.  The  composition  of  some  of  them 
is  known,  whereas  in  the  case  of  others,  we  have  no  assurance 
that  the  substances  reported  are  single  compounds  and  not  mix- 
tures of  closely  related  compounds. 

Lecithin. — Lecithin,  classed  as  a  phosphatid,  is  one  of  the 
interesting  compounds  of  this  type.  It  is  found  in  all  cells,  in 
greatest    amount    in    egg    yolk    which    contains    about    10%. 


70  PHYSIOLOGICAL    CHEMISTRY 

Lecithin  is  soluble  in  absolute  alcohol,  and  in  ether,  but  may 
be  precipitated  from  the  latter  solution  by  adding  acetone.  On 
hydrolysis  lecithin  yields  higher  fatty  acids,  glycerine,  phos- 
phoric acid  and  an  organic  base  choline.  It  is  believed  to  have 
the  following  formula,  where  R  indicates  a  fatty  acid  residue. 

0 
CH2O  —  C  —  R 

0 
CH  0  —  C  —  R 

CH2O. 

\ 

0  0  — CH2  — CH^N  (CH3)30H 

\   / 
P 

/   \ 
0  OH 

Cholesterol. — Cholesterol  is  a  substance  which  resembles  the 
fats  in  some  of  its  physical  properties,  but  has  little  relation 
to  them  chemically.  It  is  widely  distributed  in  nature,  and  is 
found  in  large  amounts  in  the  brain  and  nerve  tissue.  It  oc- 
curs also  in  small  amounts  in  the  blood,  and  in  the  bile,  from 
which  it  is  occasionally  deposited  in  gall  stones,  whence  it  is 
most  easily  obtained.  Cholesterol  is  insoluble  in  water,  acids, 
or  alkalies.  It  is  readily  soluble  in  hot  alcohol,  in  ether,  chloro- 
form, benzol,  etc.  Cholesterol  crystallizes  from  hot  alcohol  or 
other  solvents,  forming  large  colorless  plates.  It  gives  many 
color  reactions.  If  a  chloroform  solution  of  cholesterol  is  care- 
fully treated  with  concentrated  sulphuric  acid  so  as  to  form  a 
layer  of  acid  at  the  bottom  of  the  test  tube,  the  chloroform  solu- 
tion becomes  a  brilliant  red,  and  the  acid  layer  dark  red  with  a 
green  fluorescence.  This  test  is  known  as  Salkowski's  test. 
The  Lieberman-Burchard  test  is  one  of  the  best  of  the  choles- 
terol tests.     To  2  c.c.  of  a  chloroform  solution  of  cholesterol, 


FATS,    PHOSPHATIDS,    AND    ALLIED   SUBSTANCES  71 

10  drops  of  acetic  anhydride  and  2  drops  of  concentrated  sul- 
phuric acid  are  added.  A  violet  color  appears  which  quickly 
turns  to  a  blue  green. 

Cholesterol  is  an  important  substance  physiologically.  Ap- 
parently it  protects  the  red  blood  corpuscles  from  certain  harm- 
ful substances.  It  also  seems  to  exert  a  restraining  influence 
on  some  of  the  cell  enzymes,  and  thus  may  act  as  a  regulator  of 
some  of  the  cell  activities.  Cholesterol  and  certain  of  its  allied 
substances  are  important  in  giving  the  cell  its  property  of  re- 
taining the  water  which  it  contains.  The  presence  of  choles- 
terol in  the  brain  in  such  large  quantities  points  to  the  suggestion 
that  it  may  have  some  important  role  in  the  functioning  or 
properties  of  this  most  important  organ.  In  short,  there  are 
doubtless  numerous  other  roles  played  by  cholesterol  in  the  body, 
of  which  we  know  very  little  as  yet. 


CHAPTER  V 
PROTEINS 

Introductory. — The  group  of  the  protems  is  of  great  im- 
portance in  nature,  both  in  plants  and  in  animals.  Members  of 
this  group  are  found  in  every  living  cell,  and  are  necessary 
for  the  life  of  the  organism.  An  animal  may  get  on  very  well 
for  a  long  time  on  a  diet  containing  no  carbohydrate  or  fat, 
but  if  fed  on  a  diet  containing  no  protein,  the  animal  will  die. 
Plants  build  up  their  own  proteins  from  nitrogen  compounds  of 
the  soil  or  air,  but  animals  are  dependent  upon  plants  or  other 
animals  for  the  materials  out  of  which  they  construct  their 
proteins.  Plant  structures  contain  large  amounts  of  carbohy- 
drates along  with  protein  and  other  materials,  but  the  tissues 
of  animals  are  made  up  largely  of  proteins.  Thus  muscle, 
nerve,  ligament,  skin,  bone,  blood,  lymph,  hair,  nails,  feathers, 
eggs,  etc.,  all  contain  much  protein.  Often  it  is  their  chief 
solid  constituent.  In  plants  we  find  stores  of  protein  laid  away 
in  seeds,  such  as  the  grains,  and  in  many  other  places. 

Elementary  Composition. — The  members  of  the  group  of  pro- 
teins differ  very  widely  among  themselves  in  many  properties, 
but  they  are  all  alike  in  certain  respects,  as  for  example  in 
their  elementary  composition,  for  all  proteins  contain  carbon, 
hydrogen,  oxygen  and  nitrogen.  Some  contain  also  sulphur 
and  some  phosphorus.  In  addition  to  these  elements  we  find 
sometimes  others,  such  as  iron,  iodine,  copper,  manganese,  etc. 
The  proteins  show  individual  variations  in  composition,  but  an 
average  percentage  is  as  follows : 

N  16% 
S  0.3% 
P       0.4% 

72 


c 

50% 

H 

7% 

0 

22% 

PROTEINS 


73 


The  carbon  forms  the  backbone  of  the  protein  as  long  chains 
to  which  the  other  elements  are  attached.  Oxygen  is  present 
in  a  proportion  smaller  than  in  the  carbohydrates,  but  greater 
than  in  the  fats.  Nitrogen  is  present  in  various  groupings, 
chiefly  as  imid — NH —  groups  which  form  the  links  holding  the 
various  parts  of  the  protein  together ;  as  amino  groups  — NH2 ; 
and  as  — CONH2.  These  last  two  forms  represent  only  a  small 
part  of  the  total  nitrogen.  Sulphur  is  present  for  the  most 
part  in  unoxidized  form  — S —  in  one  or  two  of  the  constitu- 
ents of  the  protein  molecule,  but  may  be  present  also  in  oxidized 
form.  Phosphorus  is  present  perhaps  in  different  forms, 
chiefly  in  oxidized  form  as  phosphate. 

Olassiilcation. — A  knowledge  of  the  percentage  composition 
such  as  that  given  above  affords  us  no  clue  to  the  structure  of 
the  protein  molecule,  which  is  very  complex.  Our  knowledge  of 
the  actual  structure  of  the  proteins  is  so  limited  that  a  classi- 
fication of  the  group  has  been  worked  out  chiefly  along  other 
lines,  making  use  of  solubilities,  source,  etc.,  to  distinguish 
groups,  in  connection  with  knowledge  of  chemical  components 
so  far  as  such  information  was  available.  The  classification 
adopted  by  the  American  Society  of  Biological  Chemists  is  as 
follows : 


I. 

Simple  Proteins. 

TI 

.    Conjugated    Pro- 

III. Derived  Proteins. 

1. 

Albumins 

teins. 

A.  Primary  Protein  De- 

2. 

Globulins 

1. 

Glycoproteins 

rivatives. 

3. 
4. 
5. 
6. 

7. 

Glutelins 

Prolamines 

Albuminoids 

Histones 

Protamines 

2. 
3. 
4. 
5. 

Phosphoproteins 
Hemoglobins 
Nucleoproteins 
Lecithoproteins 

1.  Proteans 

2.  Metaproteins 

3.  Coagulated     P  r  0- 

teins 

B.     Secondary  Protein 
Derivatives 

1.  Proteoses 

2.  Peptones 

3.  Peptids 

This  classification  is  by  no  means  final  or  ideal.  Compounds 
are  known  which  do  not  fall  well  into  any  group,  whereas 
others  seem  to  be  intermediate  between  two  groups.  This  class- 
ification serves  very  well,  however,  to  bring  comparative  order 


74  PHYSIOLOGICAL    CHEMISTRY 

out  of  chaos  until  more  is  known  of  the  structural  differences 
which  differentiate  the  various  members  of  the  group.  Another 
classification,  in  use  by  English  biochemists,  differs  only  slight- 
ly from  that  given  above.  Hemoglobins  are  called  chromopro- 
teins ;  albuminoids,  scleroproteins ;  and  the  third  division  is  sub- 
divided somewhat  differently  in  the  English  classification. 

Preparation  of  Proteins  from  Materials  in  Which  They  Oc- 
cur.— Most  proteins  occur  naturally  mixed  with  a  large  number 
of  other  materials.  Only  a  few  are  found  in  a  fairly  pure 
state  in  nature.  For  purposes  of  study  it  is  desirable  to  get 
them  as  free  as  possible  from  other  compounds.  Various  meth- 
ods are  useful,  according  to  the  properties  of  the  protein  to  be 
isolated  and  those  of  the  other  substances  present  in  the  mixture 
or  tissue.  But  even  at  best  the  purification  of  a  protein  is  a 
difficult  task  and  when  finished  it  is  often  uncertain  whether 
or  not  the  substance  is  contaminated  by  some  other  protein  of 
very  similar  composition  and  properties  or  by  other  compounds. 
Plant  proteins  often  may  be  dissolved  out  with  10%  sodium 
chloride  or  with  alcohol  and  separated  from  one  another  by 
dialyzing  the  salt,  or  by  fractional  precipitation.  In  preparing 
proteins  from  animal  tissues,  the  process  is  made  somewhat 
more  difficult  by  the  fact  that  such  tissues  contain  larger 
amounts  of  autolytic  enzymes.  These  have  the  property  of 
breaking  down  the  cell  constituents  or  altering  them  if  the  tis- 
sues are  allowed  to  stand  for  some  time  before  the  proteins  are 
extracted.  Also,  it  is  difficult  to  free  the  proteins  from  the 
fatty  or  phosphatid  constituents  of  the  cell  without  altering  the 
nature  of  the  protein  itself.  Various  solvents  may  be  used,  such 
as  salt  solutions,  dilute  acids  or  alkalies.  A  method  which  is 
very  satisfactory  consists  in  freezing  the  tissue  quickly,  reduc- 
ing it  to  a  fine  powder  and  drying  while  still  frozen.  This  ma- 
terial then  may  be  extracted  with  petroleum  ether  to  remove 
fats,  phosphatids,  cholesterol,  etc.  The  residue  is  extracted  with 
10%  sodium  chloride  solution,  and  the  various  proteins  sepa- 
rated by  fractional  precipitation. 

Some  of  the  proteins  may  be  purified  by  re-crystallization. 


PROTEINS  75 

Molecular  Weig-ht. — The  molecular  weight  of  the  proteins  is 
very  high.  Estimation  of  the  molecular  weight  has  been  at- 
tended by  great  difficulties,  because  of  the  practical  impos- 
sibility of  obtaining  pure  proteins,  and  because  of  the  unstable 
nature  of  the  proteins  themselves.  The  molecular  weight  of 
these  compounds  is  so  great  that  they  cause  only  a  very  slight 
lowering  of  the  freezing  point,  so  that  this  method  of  determin- 
ing molecular  weight  has  given  results  of  questionable  reliabil- 
ity. The  boiling  point  method  cannot  be  used,  as  proteins  coag- 
ulate on  heating.  Various  other  methods  have  been  devised, 
however,  and  as  fairly  uniform  results  are  obtainable  by  differ- 
ent methods  of  analysis,  we  may  draw  fairly  accurate  conclu- 
sions as  to  the  molecular  weights  of  members  of  the  group.  One 
of  the  methods  employed  consists  in  estimating  the  sulphur  con- 
tent of  the  protein.  If  the  substance  contains  0.5%  sulphur 
then  sulphur  makes  up  1/200  of  the  protein,  and  the  molecular 
weight  will  be  200  times  that  of  the  sulphur  present.  If  there 
are  two  sulphur  atoms  in  the  protein,  they  have  a  molecular 
weight  of  64  (2X32).  The  molecular  weight  of  the  protein  will 
thus  be  200X64^12,800.  Calculated  on  this  basis  most  of  the 
proteins  will  have  a  molecular  weight  of  from  14,000-16,000. 
Calculating  the  molecular  weight  in  other  ways,  such  as  from 
the  amount  of  oxygen  some  of  the  proteins  will  take  up,  gives 
practically  identical  figures.  More  direct  methods  (measure- 
ments of  osmotic  pressure,  etc.)  confirm  these  figures,  so  that 
we  are  justified  in  assuming  the  molecular  weight  of  most  pro- 
teins to  be  roughly  in  the  neighborhood  of  15,000,  although 
some  recent  evidence  makes  it  seem  that  perhaps  these  figures, 
after  all,  are  far  too  high.  Comparing  this  figure  with  the  mole- 
cular weights  of  some  familiar  compounds  such  as  hydrochloric 
acid  (36-)-),  sodium  hydrate  (40)  and  sodium  chloride  (58+), 
we  get  an  idea  of  the  relative  hugeness  of  the  protein  molecule. 

Hydrolysis. — Any  consideration  of  the  molecular  structure 
of  compounds  having  such  enormous  molecules  would  seem  to 
present  almost  insurmountable  obstacles.  As  the  result  of  the 
work  chifly  of  Kossel,  Emil  Fischer,  Emil  Abderhalden,  and 


76  PHYSIOLOGICAL    CHEMISTRY 

Osborne,  however,  much  light  has  been  thrown  on  the  subject. 
It  has  been  found  that  proteins  if  boiled  with  concentrated  acids, 
with  alkalies,  or  if  acted  on  by  certain  enzymes  will  break  down 
ultimately  into  fairly  simple  chemical  substances.  These  are 
the  ex:  amino  acids.  On  hydrolysis,  all  proteins  are  broken 
down  into  a  mixture  of  these  compounds,  of  which  about 
twenty  have  been  obtained  from  proteins.  All  proteins,  what- 
ever their  nature,  yield  these  same  compounds.  All  the  amino 
acids  are  not  obtained  from  every  protein,  but  in  general  a  pro- 
tein yields  most  of  them.  Many  proteins  lack  one,  two,  three, 
or  perhaps  more,  and  a  few  proteins  are  made  up  of  relatively 
few  different  amino  acids.  The  analytical  methods  in  use  do 
npt  giye  products  to  the  amount  of  100%  of  the  original  pro- 
tein, for  there  are  losses  at  various  stages  in  the  process.  Usu- 
ally only  about  %  of  the  original  substance  is  accounted  for. 
From  some,  proteins  85-90%  has  been  recovered,  and  in  the 
case  of  salmine  110%,  this  apparently  impossible  result  being 
accounted  for  by  the  taking  up  of  water  when  the  amino  acid 
complexes  are  split  up.  It  is  easy  to  understand  the  causes  for 
the  difference  in  behavior  of  different  proteins.  Although  they 
all  are  made  up  of  practically  the  same  amino  acids,  these  are 
present  in  different  proteins  in  widely  varying  proportions, 
and  also  undoubtedly  are  arranged  or  put  together  differently. 
The  following  is  a  list  of  the  amino  acids  which  thus  far  have 
been  obtained  by  the  hydrolysis  of  proteins.  It  is  quite  pos- 
sible that  as  time  goes  on  others  will  be  added. 

Amino  Acids   Obtained  by  Hydrolyzing-  Protein 

A.  Monoamino — ^^monocarboxylic  acids. 

1.  GlycocoU  NH^CH.COOH 

2.  Alanine  CH3CHNHXOOH 

3.  ex  Amino  Butyric   CH,CH,CHNH.,COOH 

CH3 

\ 

4.  Valine  CH  —  CHNH,COOH 

/ 


PROTEINS 

5. 

Caprine 

CH^CHoCHg  CHoCHNH/'OOH 
CPT, 

6. 

Leucine 

\ 

CH  CHoCHNH/'OOH 

/ 

CH3 

CH3— CH., 

7. 

Isoleucine 

CH  CHNH,COOH 

/ 
CH3                  . 

CH.OH 

1 

8. 

Serine 

CHNH2 

COOH 
CH^SH 

9. 

Cystein 

CHNH2 

COOH 

CH,S  —  S  CH. 

10. 

Cystin 

CHNH2      CHNH, 

77 


COOH       COOH 
B.  Monoamino  dicarboxylic  acids. 


11.        COOH 

I 
CH, 

I 
CHNH, 

COOH 

Aspartic 


12.         COOH 

CH, 

I 
CH. 

I 
CHNH, 

I 

COOH 
Glutamic 


78 


PHYSIOLOGICAL    CHEMISTRY 


C.  Diamino-monocarboxylie   acids 
13.  Lysine  I 


NH. 


14.  Arginine 

D.  Cyclic  compounds. 

15.  Phenylalanine 


16.  Tyrosine 


17.  Tryptophane 


18.  Histidine 


CH2      CH^CH.CH^CH      COOH 

/NH3 
C  =  NH 
\NH  CH2CH2CH2CHNH2COCH 

H 
C 
HC/  \C  CH2CHNH2COOH 

HC\  /C 
C    H 
H 

H 
C 
HC/  \C  CH2CHNH2COOH 

1100X^0 
C    H 
H 

H 

C 
HC/  \C C— CH.CHNH^COOH 

HC\/C\/C 
C         N    H 
H        H 

H 

C  — N 


\ 

/ 

C  — N 

I         H 
CHo 


CH 


CHNH2 
COOH 


PROTEINS  79 

H.C  —  CH2 

'I        I 
]».  Proline  H^C       C— COOH 

\/    H 

N 
H 

(OH) 
HgC  —  C  H 

I        I 
20.  Oxyproline  H^C       C.COOH 

\/ 

N 

H 

The  position  of  the  hydroxyl  is  uncertain. 

General  Properties  and  Reactions  of  Amino  Acids. — Solubil- 
ity, Taste,  Optical  Activity. — The  amino  acids  obtained  by 
hydrolysis  of  protein  are  crystalline  substances,  which  are 
readily  soluble  in  water,  with  the  exception  of  cystine,  which 
dissolves  with  difficulty  in  both  cold  and  hot  water,  and  of 
tyrosine,  which  is  quite  insoluble  in  cold  water,  but  dissolves 
more  readily  in  hot  water.  Solutions  of  monoamino-monocar- 
boxylic  acids  are  neutral  in  reaction.  Solutions  of  dicarboxylic 
acids  are  acid,  and  of  diamino  acids  are  alkaline.  The  solu- 
tions of  monoamino-monocarboxylic  acids  in  reality  have  both 
acid  and  basic  properties  and  should  properly  be  classed  as  am- 
photeric. They  all  dissolve  in  dilute  acids  or  alkalies,  except 
cystin,  which  is  not  readily  soluble  in  dilute  ammonia.  The 
amino  acids  vary  in  taste.  Glycoeoll,  alanine  and  caprine  are 
sweet,  leucine  is  tasteless  and  isoleucine  is  bitter.  With  the 
exception  of  glycoeoll,  all  the  amino  acids  are  optically  active 
and  exist  in  two  forms,  a  dextro-  and  a  levorotatory.  Usually 
only  one  of  the  two  is  found  as  a  protein  constituent,  and  this 
is  more  often  the  levorotatorj^  variety.  If  proteins  are  broken 
down  by  hydrolysis  with  alkali,  the  resulting  amino  acids  are 
racemic,  that  is,  they  exist  as  equal  amounts  of  the  two  optical 
isomers.  The  form  of  the  acids  not  present  in  the  protein  is 
believed  to  be  produced  during  the  hydrolysis.     Hydrolysis  by 


80  .  PHYSIOLOGICAL    CHEMISTRY 

enzymes,  and  to  a  certain  extent  by  acids  produces  optically 
active  acids,  either  dextro-  or  levorotatory,  but  does  not  cause 
raeemization. 

Acids,  Bases. — The  amino  acids  add  on  acids  such  as  hydro- 
chloric at  the  amino  group.    Thus 

H 

/ 

—  NH.-,  4-  HCl  -^  —  N  =  H, 

\ 
CI 

The  acid  raises  the  valence  of  the  nitrogent  to  five.  They  also 
interact  with  bases  to  form  salts. 

—  COOH  +  NaOH  -^  —  COONa  +  H^O 

At  the  amino  group  the  amino  acids  add  on  the  salts  of  certain 
metals  such  as  cupric  chloride,  mercuric  chloride,  etc.  'This 
property  is  often  made  use  of  to  precipitate  amino  acids. 

Formaldehyde. — With  formaldehyde,  amino  acids  form  meth- 
ylene compounds.  The  basic  properties  of  the  amino  group  are 
thus  greatly  reduced,  and  the  terminal  carboxyl  group  can  be 
titrated  with  a  standard  alkali.  On  this  process  a  much  used 
method  for  estimating  amino  acids  is  based. 

!  ! 

HC  — NH, +  H,CO^  HC  — N  =  CH2  +  H.O 

COOH  COOH 

Carhamino  Reaction. — The  amino  acids  interact  with  carbon 
dioxide  in  the  presence  of  calcium  salts  to  form  carbamino  com- 
pounds.    These  have  the  following  structure: 

R  _  CH  —  NH  —  C  =  0 

1  I 

0  =  C  —  0  —  Ca  —  0 

If  the  nitrogen  in  this  compound  is  determined,  and  also  the 
amount  of  CO.,  which  is  combined,  a  relationship  between  the 


PROTEINS  81 

amounts  of  nitrogen  and  CO2  may  be  established.    In  case  the 

CO 
radicle  R  above  contains  no  nitrogen  — -^  ==  1  for  the  above  com- 

N 

pound.  If  the  radicle  R  contains  nitrogen,  however,  as  will  be 
the  case  if  the  compound  contains  diamino  acids,  or  is  a  sub- 
stance knoAvn  as  a  peptid,  in  which  two  or  more  amino  acids  are 

CO 
linked  together,  ^r—  will  be  less  than  1. 

Oxidation. — Oxidation  of  amino  acids  may  yield  a  variety  of 
products  according  to  the  strength  of  the  oxidizing  agent.  The 
NHo  groups  are  not  split  off  by  acids  or  by  alkalies  to  any  ex- 
tent. Alkalies,  however,  split  arginine  into  ornithine  and 
urea,  and  split  off  the  sulphur  from  cystin  and  cystein.  The 
small  amount  of  ammonia  given  off  in  acid  hydrolysis  of  pro- 
tein is  believed  to  come  from  the  few  acid  amid  groups 
— CO — NH2  present.  Oxidation  with  permanganate  or  hydro- 
gen peroxide  yields  pyruvic  acid  from  alanine.  This  compound 
is  interesting  since  it  is  believed  to  be  one  of  the  steps  in  the 
breaking  down  of  carbohydrates  in  the  body,  and  thus  may  be 
a  substance  by  way  of  which  amino  acids  and  monosaccharides 
can  be  converted  into  one  another  in  the  body.  The  substance 
has  the  formula 

CH3 

I 
C  =  0 

I 
COOH 

Nitrous  Acid. — ^With  nitrous  acid  the  oc  amino  acids  are 
broken  down.  Their  nitrogen  is  liberated  in  the  form  of  the 
free  gas.  This  will  be  recognized  as  the  familiar  reaction  of 
nitrous  acid  with  primary  amines. 

R  NH2  +  HONO  -^  ROH  +  N,  +  H^O 

This  reaction  is  the  basis  for  the  Van  Slyke  method  for  estimat- 
ing amino  acids,  a  method  which  has  proven  most  useful  in 
helping  to  settle  some  of  the  difficult  questions  with  reference 
to  the  fate  of  the  proteins  in  the  body.     From  this  brief  re- 


82  PHYSIOLOGICAL   CHEMISTRY 

view  of  some  of  the  important  facts  relating  to  the  amino  acids 
we  will  turn  our  attention  to  the  general  properties  and  reac- 
tions of  the  proteins  themselves. 

General  Protein  Reactions 

The  general  protein  tests  may  be  divided  into  two  groups, 
the  color  tests  and  the  precipitation  tests. 

Color  Tests. — The  protein  color  tests  are  by  no  means  specific 
for  proteins.  They  are  tests  which  are  given  by  certain  group- 
ings or  certain  constituents  generally  found  in  the  protein  mole- 
cule. If  the  gi'ouping  upon  which  a  test  depends  is  absent 
from  a  particular  protein,  that  protein  will  not  respond  to  the 
test  in  question.  Thus  a  single  positive  test  should  not  be 
taken  as  evidence  of  the  presence  of  a  protein,  but  should  be 
confirmed  by  some  other  test. 

Biuret  Test. — If  concentrated  sodium  hydroxide  is  added  to 
a  protein  solution,  and  then  a  few  drops  of  very  dilute  copper 
sulphate  solution,  a  violet  color  appears  either  at  room  tem- 
perature or  on  slight  warming.  This  test  is  named  from  the 
fact  that  it  is  given  by  a  substance  biuret  which  is  made  from 
two  molecules  of  urea  with  loss  of  NH3 

NH, 

\ 

NH2  C  =  0 

/  / 

2  CO  ->       HN 

\  \ 

NH,  C  =  0 

/ 

Biuret 

This  substance  does  not  occur  as  a  constituent  of  the  protein 
molecule.  The  test  depends  on  the  presence  of  two  amid 
groups  — CO  — NH2  united  either  directly  or  by  a  carbon  or 
nitrogen  atom.  One  of  the  amino  groups  may  be  substituted 
as  — CO— NHR  but  not  both  of  them.    If  the  NH2  of  the  amid 


PROTEINS  83 

groups  is  split  off  by  the  action  of  strong  acids,  the  proteins  will 
no  longer  give  a  biuret  reaction.  Ammonium  salts  such  as  am- 
monium sulphate  interfere  with  the  test,  and  should  be  decom- 
posed by  boiling  the  mixture  with  strong  alkali  before  making 
the  test.  Some  of  the  proteins,  e.g.,  the  histones,  and  some  of 
the  products  of  protein  hydrolysis  give  a  reddish  color. 

Millon's  Test.- — If  a  few  drops  of  Millon's  reagent  (a  solution 
made  by  dissolving  mercury  in  concentrated  nitric  acid)  is 
added  to  a  protein  solution,  a  yellowish  preciptate  forms.  On 
boiling,  this  precipitate  turns  rose  pink.  If  the  boiling  is  con- 
tinued the  pink  color  is  destroyed  and  the  precipitate  turns 
brown.  At  times  the  whole  solution  becomes  pink.  If  the 
protein  happens  to  be  insoluble,  it  will  give  the  test  quite  as 
well,  turning  a  very  decided  pink  or  red.  The  test  depends  on 
the  presence  in  the  protein  of  tyrosine,  and  is  given  only  by 
those  proteins  which  contain  this  amino  acid.  Phenol  or  any 
other  compound  which  contains  a  hydroxyphenyl  group  will 
give  the  test,  however.  The  dihydroxybenzenes  do  not  give  the 
test  unless  one  hydroxyl  group  is  substituted.  Chlorides,  alco- 
hol or  hydrogen  peroxide  will  interfere  with  the  test.  Thus  it 
is  not  serviceable  to  test  urine  for  protein,  since  urine  contains 
large  amounts  of  sodium  chloride. 

Xanthoproteic  Test. — If  a  protein  is  warmed  with  concen- 
trated nitric  acid  the  mixture  turns  lemon  yellow.  On  the  ad- 
dition of  alkali  the  color  changes  to  a  deep  orange.  The  pro- 
tein need  not  be  in  solution.  Students  of  chemistry  will  recall 
having  performed  this  test  upon  themselves  by  getting  con- 
centrated nitric  acid  upon  the  fingers,  producing  the  familiar 
yellow  spots.  The  skin  is  made  of  protein  material.  This  test 
depends  upon  protein  constituents  containing  the  benzene  ring, 
namely  tyrosine,  phenylalanine  and  tryptophane.  The  colored 
substance  is  a  nitro  derivative  of  benzene.  Any  substance,  pro- 
tein or  otherwise,  containing  a  benzene  ring  will  respond  to 
this  test. 

Adamkiewicz  or  Hopkins-Cole  Test. — If  glacial  acetic  acid, 
or  a  solution  of  glyoxylic  acid   (prepared  by  reducing  oxalic 


84  PHYSIOLOGICAL    CHEMISTRY 

acid  with  magnesium  or  sodium  amalgam)  is  added  to  a  pro- 
tein solution,  and  concentrated  sulphuric  acid  added  so  as  to 
form  a  layer  at  the  bottom  of  the  test  tube,  a  violet  ring  will 
form  at  the  juncture  of  the  two  liquids.  This  test  is  due  to  the 
tryptophane  in  the  protein  molecule,  and  only  those  proteins 
containing  this  amino  acid  will  respond  to  the  test.  When 
glacial  acetic  acid  is  used,  the  test  is  believed  to  be  due  to  the 
presence  as  an  impurity  of  glyoxylic  acid,  HCO — COOH,  or 
other  aldehydes. 

Sulphur  Test. — If  concentrated  sodium  hydroxide  and  lead 
acetate  are  added  to  a  protein  solution  and  the  mixture  boiled, 
a  brown  or  black  color  appears.  The  unoxidized  sulphur  of 
the  cystein  or  cystin  is  split  off  and  combines  with  the  lead  to 
form  the  dark  brown  or  black  lead  sulphide. 

Precipitation  Reactions.  Colloids. — Proteins  may  be  pre- 
cipitated by  a  variety  of  reagents.  The  behavior  of  protein  so- 
lutions with  precipitation  reagents,  and  in  fact  many  other 
properties  of  protein  solutions  indicate  that  the  proteins  do 
not  form  true  solutions  such  as  that  of  sodium  chloride  in  water. 
They  form  what  are  known  as  colloidal  solutions.  Substances 
of  this  nature  are  called  colloids.  The  particles  of  a  substance 
in  colloidal  solution  are  so  large  that  they  will  not  pass  through 
the  pores  of  a  parchment  membrane.  The  group  was  named 
"colloids"  by  Graham  to  distinguish  these  compounds  from 
substances  which  form  a  "true  solution,"  to  which  the  name 
crystalloids  was  applied.  Crystalloids  in  solution  are  divided 
into  such  minute  particles  that  they  will  pass  through  the  pores 
of  a  parchment  membrane.  As  a  matter  of  fact,  colloids  often 
crystallize,  but  as  a  rule  less  readily  than  crystalloids. 

Classification  and  Properties  of  Colloids. — The  colloids  are 
by  no  means  a  unified  chemical  group,  for  substances  of  the 
most  widely  diverse  chemical  nature,  such  as  metals,  salts,  acids, 
bases,  proteins,  carbohydrates,  etc.,  may  form  colloidal  solu- 
tions. The  term  "colloidal"  refers  in  fact  to  a  state  of  mat- 
ter, and  not  to  a  class  of  compounds.  Many  substances  of  the 
greatest  biological  importance  form  colloidal  solutions,  in  fact 


I  PROTEINS  85 

the  constituents  of  the  living  cell  are  believed  to  be  in  a  col- 
loidal state,  so  that  the  properties  of  colloids  are  both  inter- 
esting and  important.  The  group  is  divided  into  two  classes, 
hydrophile  or  emulsoid  colloids,  and  suspensoid  colloids.  Hy- 
drophile  colloids  more  closely  approach  the  crystalloids  in  their 
properties,  whereas  suspensoid  colloids  are  more  nearly  like 
suspensions.  As  a  matter  of  fact  there  is  no  sharp  dividing 
line  either  between  the  groups,  or  between  emulsoids  and  true 
solutions,  or  suspensoids  and  suspensions,  since  all  gradations 
existr*  Peptones,  although  belonging  to  the  class  of  derived  pro- 
teins, will  pass  through  a  parchment  membrane  fairly  well,  and 
are  thus  between  the  emulsoids  and  crystalloids.  Certain  metal 
hydroxides  form  gels,  but  are  precipitated  easily  by  electro- 
lytes, and  are  thus  between  the  emulsoids  and  suspensoids. 
Kaolin  shaken  up  with  water  is  midway  between  the  suspensoids 
and  true  suspensions. 

Emulsoid  colloids  are  characterized  by  the  fact  that  they 
form  gels  if  sufficiently  concentrated,  and  are  not  easily  pre- 
cipitated by  the  addition  of  salts.  If  in  solution,  the  substance 
is  called  a  hydrosol,  if  in  a  gel  form,  a  hydrogel.  Examples  of 
emulsoids  are  albumin,  gelatine  and  other  proteins,  starch,  etc. 
The  emulsoid  colloids  have  some  attraction  for,  or  relation  with 
the  water  surrounding  them.  This  property  is  of  the  greatest 
importance,  for  the  colloids  of  the  living  protoplasm  aid  in 
holding  the  water  which  is  essential  to  the  life  and  functioning 
of  living  cells. 

The  suspensoid  colloids  do  not  form  gels,  and  are  easily  pre- 
cipitated by  the  addition  of  even  a  small  amount  of  a  salt.  Ex- 
amples of  this  class  are  colloidal  metals,  sulphides,  etc.  They 
seem  to  have  little  relation  to  the    water    surrounding    them. 

The  particles  in  colloidal  solutions  are  so  small  that  they  will 
pass  through  an  ordinary  filter  with  ease.  Filters  impregnated 
with  collodion  have  been  prepared,  however,  by  means  of  which 
colloid  particles  can  be  held  back.  In  this,  and  other  ways,  the 
size  of  the  particles  has  been  estimated.     The  size  of  colloidal 


86  PHYSIOLOGICAL    CHEMISTRY 

particles  runs  from  1-130/x/i,,  one  /x/i,  being  the  millionth  part 
of  a  millimeter. 

Tyndall's  Phenomenon. — Colloidal  solutions  show  an  inter- 
esting behavior  known  as  Tyndall's  phenomenon.  If  a  beam 
of  light  is  passed  through  a  colloidal  solution,  the  path  of  the 
ray  becomes  visible,  in  much  the  same  manner  as  the  path  of  a 
ray  of  sunlight  in  a  dusty  room.  The  light  is  dispersed  or  re- 
flected from  the  particles  of  the  colloid. 

Electrical  Properties  of  Colloids. — Colloidal  particles  carry 
electrical  changes  just  as  ions  are  electrically  charged.  This 
may  be  demonstrated  by  passing  an  electric  current  through  a 
colloidal  solution.  The  particles  of  the  colloid  will  move  to  the 
positive  or  negative  pole  according  to  the  nature  of  the  charge 
carried, — a  colloid  with  a  negative  charge  travelling  to  the 
positive  pole,  and  vice  versa.  This  phenomenon  is  known  as 
catajihoresis.  Whereas  some  colloidal  particles  probably  have 
but  one  electrical  charge,  undoubtedly  they  often  carry  more 
than  one.  A  protein  in  colloidal  solution  will  have  a  positive 
charge  if  the  solution  is  acid  in  reaction,  but  a  negative  charge 
if  the  solution  is  alakaline.  We  may  imagine  that  this  is 
brought  about  as  follows :  in  acid  solution  the  protein  combines 
with  some  of  the  acid,  for  example  hydrochloric  acid.  From 
this  complex  compound  negatively  charged  chlorine  ions  are 
given  off  into  the  water,  and  positive  charges  will  remain  on 
the  colloid  particles.  In  alkaline  solution  the  protein  forms 
salts,  such  as  the  sodium  salt.  Sodium  ions  are  given  off,  carry- 
ing positive  charges,  and  negative  charges  will  remain  on  the 
colloid  particles.  These  facts  are  of  great  importance  in  many 
of  the  precipitation  reactions  of  the  colloids. 

Methods  of  Precipitating-  Colloids. — Some  colloidal  solutions 
will  precipitate,  the  colloid  flocking  out,  merely  on  standing. 
Some  will  precipitate  if  they  are  boiled.  Some  substances  are 
soluble  in  hot  water,  but  their  solutions  will  solidify  on  cooling. 
Some  colloids  are  thrown  out  of  solution  by  the  addition  of  an 
electrolyte.  The  suspensoid  colloids  are  precipitated  by  adding 
a  very  small  amount  of  an  electrolyte  such  as  a  salt  or  an  acid, 


PROTEINS  87 

but  the  emulsoid  colloids  are  precipitated  much  less  readily, 
that  is,  only  by  adding  much  more  of  the  electrolyte.  It  has 
been  observed  that  the  effective  part  of  the  precipitating  salt  or 
substance  is  the  ion  bearing  the  opposite  charge  to  that  on  the 
colloid.  If  the  precipitating  part  of  the  salt  is  the  metal,  then 
in  general  colloids  bearing  negative  charges  will  be  precipitated. 
In  this  connection  it  has  been  observed  that  trivalent  metals  are 
better  preciptation  reagents  than  divalent  metals,  and  divalent 
metals  in  general  are  better  precipitation  reagents  than  mono- 
valent metals.  Thus  to  precipitate  a  given  colloid  from  solu- 
tion a  ferric  salt  is  better  than  a  mercuric  salt,  and  a  mercuric 
salt  better  than  a  sodium  salt.  That  is,  a  smaller  concentration 
of  ferric  chloride  than  of  mercuric  chloride  is  required,  etc.  But 
all  ions  of  the  same  valency  do  not  have  equal  precipitation 
powers.     They  vary  according  to  their  solution  tension. 

"When  a  colloid  is  precipitated  by  an  electrolyte  the  precipi- 
tate contains  some  of  the  precipitating  ion,  so  the  precipitate  is 
believed  to  be  a  compound  of  the  colloid  and  the  precipitating 
ion.  The  precipitation  of  colloids,  however,  is  undoubtedly  de- 
pendent on  other  and  more  complicated  factors  than  the  mere 
formation  of  salts  or  similar  compounds  of  the  colloids.  For 
further  discussion  of  this  subject  the  student  is  referred  to 
larger  or  more  specialiezd  works. 

A  suspensoid  colloid,  as  has  been  stated,  is  easily  precipitated 
by  the  addition  of  an  electrolyte.  If  a  small  amount  of  an  emul- 
soid colloid  is  added  to  a  suspensoid  colloid  solution,  the  latter  is 
much  less  easily  precipitated.  The  suspensoid  colloid  is  "pro- 
tected" by  the  emulsoid  colloid  (albumin,  for  example).  This 
phenomenon  is  called  the  protective  action  of  colloids. 

The  properties  of  colloidal  solutions  are  being  investigated 
at  present  by  many  scientific  men. 

Returning  to  the  methods  of  precipitating  proteins  in  particu- 
lar, we  find  a  variety  of  methods  available. 

Heat. — Many  proteins  are  precipitated  by  heat.  A  slightly 
acid  reaction,  and  the  presence  of  salts  is  desirable  if  precipi- 
tation is  to  be  complete.    Alkaline  solutions  of  proteins  do  not 


88  PHYSIOLOGICAL    CHEMISTRY 

precipitate  on  boiling.  Neutral  solutions,  especially  if  salts 
have  been  removed  by  dialysis,  will  precipitate  only  imperfect- 
ly. Solutions  of  some  proteins,  i.e.,  casein,  gelatine  and  sec- 
ondary derived  proteins  such  as  proteoses  and  peptones  do  not 
precipitate  on  boiling.  In  general  for  each  protein  there  is  a 
specific  precipitation  temperature,  but  experimental  conditions 
such  as  the  amount  of  salts  present,  the  rapidity  of  heating  and 
other  factors  cause  rather  wide  variations  in  the  precipitation 
temperature.  A  protein  precipitated  by  boiling  in  weak  acid 
solution  cannot  readily  be  redissolved.  Some  as  yet  unknown 
change  has  taken  place  in  the  protein  molecule,  and  the  sub- 
stance is  said  to  be  coagulated.  Proteins  may  be  coagulated 
also  in  other  ways. 

Mineral  Acids. — Proteins  are  precipitated  by  the  addition 
of  small  amounts  of  the  strong  mineral  acids, — hydrochloric, 
sulphuric  and  nitric.  The  precipitate  dissolves  in  excess  of  the 
acid,  particularly  if  the  solution  is  heated.  Glacial  acetic  acid 
does  not  precipitate  proteins.  There  has  been  much  discussion 
of  the  nature  of  this  precipitation.  Probably  the  proteins  are 
thrown  down  in  the  form  of  salts.  Precipitation  with  concen- 
trated nitric  acid  is  often  used  as  a  test  for  proteins.  To  the 
solution  to  be  tested,  concentrated  nitric  acid  is  added  carefully 
so  that  it  will  form  a  layer  at  the  bottom  of  the  test  tube.  In 
the  presence  of  protein  a  cloudy  ring  appears  at  the  juncture  of 
the  two  liquids. 

Salts. — Proteins  are  precipitated  by  salts.  The  salts  of 
heavy  metals,  such  as  copper,  iron,  mercury,  lead,  etc.,  will 
throw  down  the  proteins  from  their  solutions.  The  precipi- 
tates formed  are  in  many  cases  true  salts  of  the  protein  and 
the  metal.  "Where  this  is  the  case,  the  precipitation  takes  place 
best  usually  in  a  weakly  alkaline  solution,  for  in  this  condition 
the  proteins  are  negatively  charged,  and  will  combine  with  the 
positive  metal  ions.  Some  proteins,  such  as  protamines  and  his- 
tones,  which  have  large  amounts  of  dianiino  acds,  form  alkaline 
solutions  and  the  protein  caries  positive  charges.  More  alkali 
must  be  added  to  give  .these  proteins  negative  charges  than  in 


PROTEINS  89 

the  ease  of  albumins.  Casein,  which  contains  much  dicarboxylic 
acid  (glutamic)  carries  negative  charges  (since  it  gives  off 
hydrogen-ions  to  the  water).  Casein  may  thus  be  precipitated 
by  metals  even  in  slightly  acid  solution. 

The  conditions  governing  precipitation  of  proteins  by  metals 
are  somewhat  complicated  by  the  fact  that  some  metals  such 
as  mercury,  gold,  copper,  and  others  (these  metals  have  a  lower 
solution  tension  than  hydrogen)  combine  with  the  amino  group 
also,  so  that  salts  of  these  metals  will  precipitate  proteins  in 
weakly  acid  as  well  as  in  weakly  alkaline  solution. 

Three  salts  much  used  to  precipitate  proteins  are  ammonium 
sulphate,  magnesium  sulphate  and  sodium  chloride.  High  and 
varying  concentrations  of  these  salts  are  necessary  to  throw 
down  the  different  proteins.  By  the  use  of  suitable  amounts 
of  these  salts  some  of  the  protein  groups  may  be  separated  from 
others.  The  process  is  known  as  "salting  out."  The  proteins 
are  not  coagulated,  and  may  be  redissolved  on  removal  of  the 
salt. 

Alkaloidal  Reagents. — Many  compounds  known  as  alkaloidal 
reagents  will  precipitate  proteins.  Among  these  are  several 
acids  such  as  tannic,  picric,  phosphotungstic,  phosphomolybdic, 
ferrocyanic,  chromic,  and  dichromic.  The  precipitates  undoubt- 
edly are  compounds  of  protein  with  the  negative-ion  of  the  pre- 
cipitating reagent.  The  tests  are  thus  carried  out  to  best  ad- 
vantage in  weakly  acid  solution.  Exceptions  to  this  statement 
depend  upon  conditions  similar  to  those  discussed  under  pre- 
cipitation by  the  addition  of  salts. 

Alcohol. — Alcohol  in  sufficient  concentration  will  precipi- 
tate many  of  the  proteins.  Some  few  are  soluble  in  alcohol, 
however.  If  the  precipitate  is  allowed  to  stand  in  the  alco- 
hol, it  will  become  coagulated,  and  cannot  be  redissolved  on  re- 
moval of  the  alcohol. 

Structure  of  the  Protein  Molecule 

Since  proteins  make  up  so  large  a  part  of  living  tissue,  and 
are  indispensable  to  life  it  would  be  of  great  interest  to  find  out, 


90  PHYSIOLOGICAL   CHEMISTRY 

how  the  protein  molecule  is  constructed.  This  problem  has  been 
studied  by  some  of  the  foremost  biochemists  for  a  long  time,  and 
although  the  formula  for  a  protein  is  still  unknown,  much  is 
now  known  as  to  the  manner  in  which  the  parts  of  a  protein 
molecule  are  put  together.  On  hydrolysis,  proteins  yield  a  mix- 
ture of  amino  acids.  These,  then,  must  be  the  building  stones 
out  of  which  the  proteins  are  constructed.  How  are  these 
building  stones  put  together?  Much  evidence  has  accumulated 
to  show  that  the  amino  acids  are  joined  together  in  long  chains, 
the  different  units  being  linked  by  what  is  known  as  the  amid  or 
"peptid"  linking, — the  union,  with  loss  of  water,  of  the  ear- 
boxyl  group  of  one  acid  with  the  amino  group  of  another. 

NH2CH2COOH  +  NH2CH2COOH  -^ 
NH2CH2CO  —  NH  —  CH2COOH  +  H2O 

A  compound  of  this  type  is  called  a  peptid, — if  made  from  two 
amino  acids,  a  dipeptid,  if  from  three  amino  acids,  a  tripeptid, 
if  from  many  amino  acids,  a  polypeptid.  On  partial  hydrolysis 
of  proteins,  such  compounds  actually  have  been  found  in  the 
resulting  mixture,  and  have  been  shown  to  be  identical  with 
compounds  of  known  structure  built  up  in  the  laboratory.  The 
most  complex  compound  of  this  nature  yet  synthesized  was 
prepared  by  Emil  Fischer.  He  prepared  a  substance  having 
eighteen  amino  acids  in  the  chain,  thus  an  octadecapeptid.  This 
compound  had  a  molecular  weight  of  1213,  and  from  its  prop- 
erties, was  still  much  simpler  than  a  protein.  The  synthesis  of 
such  compounds  is  extremely  laborious  and  expensive,  so  that 
no  attempt  has  been  made  to  carry  the  process  further.  One  of 
the  factors  tending  to  increase  the  labor  of  synthesis  is  the  fact 
that  the  amino  acids  used  are  optically  active.  As  ordi- 
narily obtained,  they  consist  of  equal  portions  of  the  dextro- 
and  levorotatory  forms,  and  must  be  separated  into  the  two 
varieties.  The  method  most  used  to  accomplish  this  consists 
in  preparing  the  salt  of  the  two  forms  with  some  optically  ac- 
tive base,  such  as  brucine,  cinchonine,  etc.  The  brucine  com- 
pounds of  the  d-  and  1-forms  may  be  separated  since  they  usu- 


PROTEINS  91 

ally  vary  considerably  in  solubility,  and  on  concentration  one  or 
the  other  will  crystallize  out  first.  A  second  method  consists 
in  allowinjy  various  microorganisms  to  act  on  the  mixture  of 
the  two  optical  isomers.  Usually  one  form  will  be  destroyed 
by  the  microorganism,  the  other  to  a  much  smaller  degree,  or 
not  at  all. 

The  optically  active  acids  then  may  be  built  up  into  peptids 
at  will.    Three  methods  are  in  use  to  effect  this  synthesis. 

1.  Ester  metliod. — 

Glycocoll  ester  is  converted  into  a  ring  compound 

C2H3O  0  C  CH2NH2    ] 


+                       ^    2C,H,0: 

H  + 

,N  CH2COOC2H5 

CH2  —  NH 

/                     \ 

CO                           CO 

\          / 

NH  —  CH, 

Under  the  action  of  alkali  this  ring  compound  is  split  open, 
forming  a  dipeptid 

NH2CH2CO  NH  CH2COOH 

Of  course  the  amino  acids  used  may  be  varied,  and  thus  differ- 
ent dipeptids  obtained.  The  method  serves  only  to  build  up 
dipeptids,  however,  and  if  two  different  amino  acids  are  used 
it  has  certain  other  disadvantages. 

2.  Syntliesis  hy  means  of  acid  chlorides. — 

If  glycocoll  is  treated  with  chloracetyl-chloride  the  following 
reaction  takes  place: 

CICH2CO       CI  +  NH0CH2COOH  -^  HCl  + 
CI  CH2CO  —  NH.  CH2COOH 

By  the  action  of  ammonia,  the  chlorine  atom  may  be  replaced 
by  NH2,  and  a  dipeptid  results. 

NH,CH,CO  — '  NH.  CHXOOH. 


92  PHYSIOLOGICAL    CHEMISTRY 

This  may  be  treated  with  ehloracetyl  chloride  again,  and  in  a 
manner  perfectly  analagous  to  the  first  reaction,  a  tri-peptid 
or  higher  peptid  built  up.  Here  again  various  amino  acids,  and 
various  halogen  derivatives  may  be  used.  The  limitation  of  this 
method  lies  in  the  fact  that  the  chain  can  be  extended  only  at 
one  end. 

3.  By  treating  an  amino  acid  or  a  peptid  witJi  pJiospTiorus 
pentachloride,  the  earboxyl  group  is  converted  into  an  acid 
chloride  group  which  then  may  be  caused  to  interact  with  the 
amino  group  of  an  amino  acid  or  a  peptid  in  a  manner  analagous 
to  that  described  in  the  second  method  of  synthesis.  Thus  gly- 
cocoU  will  be  converted  into  NH2CH0CO  CI  which  may  be  com- 
bined with  a  second  glycocoll  molecule  or  other  amino  acid  to 
form  a  dipeptid  as  in  2.  , 

The  compounds  prepared  in  these  ways  show  many  of  the 
reactions  characteristic  of  the  products  of  partial  hj^drolysis  of 
proteins, — thus  some  give  the  biuret  reaction,  those  containing 
tyrosine  give  the  Millon  test,  those  containing  tryptophone  give 
the  Adamkiewicz  test,  and  certain  of  the  more  complex  of  them 
are  precipitated  by  some  of  the  protein  precipitants.  Many  of 
them  even  are  split  by  enzymes  found  in  the  body  which  are 
known  to  split  protein  decomposition  products.  These  synthetic 
products  thus  are  identical  with  those  obtained  in  protein  hy- 
drolysis, a  fact  which  is  one  of  the  strongest  pieces  of  evidence 
that  the  proteins  are  constructed  on  the  general  plan  of  the 
polypeptids.  Other  evidence  for  this  form  of  linkage  in  the 
protein  is  given  by  various  facts.  The  proteins  react  only  to  a 
very  limited  extent  with  reagents  which  interact  with  free  NH2 
groups.  Very  little  hydrochloric  acid  is  taken  up  by  them,  the 
formaldehyde  reaction  described  above  shows  relatively  few 
free  amino  groups,  as  does  also  the  Van  Slyke  method  also  de- 
scribed above.  In  alkaline  hydrolysis  the  amino  acids  become 
racemic  for  the  most  part.  Only  those  whose  earboxyl  groups 
are  combined,  as  is  the  case  in  the  amid  linking,  will  become 
racemic  on  hydrolysis.     All  these  results  are  explained  if  the 


PROTEINS  93 

amino  acids  making  up  the  proteins  are  assumed  to  be  united 
by  the  "peptid  linking." 

The  behavior  of  polypeptids  with  enzymes  is  especially  in- 
teresting, and  demonstrates  the  extremely  specific  character  of 
enzyme  action.  For  example,  it  has  been  shown  that  the  di- 
peptid  d-alanyl-d-alanine  is  split  by  pancreatic  juice,  but  not 
d-alanyl-1-alanine.  In  general,  those  peptids  made  up  of  the 
optical  form  of  the  amino  acids  which  is  found  in  nature  will 
be  split  by  enzymes.  Peptids  made  from  the  optical  isomers 
which  do  not  occur  in  nature  are  not  split,  or  are  split  less 
readily  by  enzymes. 

From  a  study  of  the  polypeptids  it  has  been  learned  that  the 
ease  with  which  a  given  polypeptid  may  be  precipitated  from 
solution  depends  not  only  on  the  size  of  the  molecule,  but  also 
upon  the  particular  amino  acids  present  in  it.  Thus  a  poly- 
peptid containing  tyrosine,  tryptophane  or  cystin  will  pre- 
cipitate at  a  much  lower  concentration  of  a  given  precipitating 
reagent  than  a  second  polypeptid  of  equal  molecular  weight, 
containing  none  of  these  acids.  Many  polypeptids,  both  those 
prepared  synthetically  and  those  obtained  from  protein  hydro- 
lysis will  give  characteristic  protein  reactions,  though  no  poly- 
peptid has  been  prepared  which  closely  approaches  the  pro- 
teins in  the  complexity  and  size  of  its  molecule.  , 

On  hydrolizing  a  protein,  that  is,  breaking  it  down  into  sim- 
pler products,  the  process  evidently  is  not  a  symmetrical  divi- 
sion and  redivision  of  the  molecule.  In  tryptic  digestion  tyro- 
sine, tryptophane  and  cystine  are  split  off  much  quicker  than 
alanine,  leucine  and  valine,  whereas  glycocoll,  proline  and 
phenylalanine  are  not  split  off  even  after  prolonged  digestion. 
We  must  assume,  therefore,  that  certain  groups  or  "building 
stones, ' '  are  split  off  from  the  great  protein  moleeule,  which  is 
thus  reduced  through  various  stages  to  proteoses,  peptones  and 
in  complete  hydrolyss  to  the  amino  acids. 

Putrefaction  of  Proteins. — AVhen  proteins  are  broken  down 
by  the  action  of  bacteria  in  the  intestines,  products  are  formed 
some  of  which  are  very  injurious  to  the  organism.     The  amino 


94 


PHYSIOLOGICAL    CHEMISTRY 


acids  produced  by  the  hydrolysis  of  the  proteins  are  attacked 
and  partially  broken  down  in  a  variety  of  ways.  From  trypto- 
phane by  the  removal  of  its  side  chain,  skatol  and  indol  are 
produced. 

H  H 

C  C 

//\  //\ 

HC      C  — CCH,  HC      C  — CH 


HC      C      CH 

C       N 
H      H 
Skatol 


HC      C  —  CH 

C       N 
H      H 
Indol 


From  amino  acids,  by  removal  of  COg  from  the  carboxyl  group 
various  amines  are  produced,  e.g.,  from  tyrosine,  tyramine 


H 
C 

/   \ 
HC  C  CH2 

I  II 

HO  C  C  H 

\   / 
C 
H 


CH  NH,  COOH        HC 


H 
C 

/   \ 


C  CH,  CH,NH, 


HO 


C  CH 

\   / 
C 
H 


From  histidine,  histamine  is  produced  in  a  similar  way.  Such 
compounds  are  known  by  the  name  "ptomaines,"  and  they  are 
extremely  toxic.  Cystine  and  cystein  give  rise  to  hydrogen  sul- 
phide and  other  sulphur  compounds.  A  condition  of  constipa- 
tion, in  which  intestinal  contents  are  retained  unduly  long  in 
the  body,  and  putrefaction  thus  prolonged,  favors  the  produc- 
tion of  such  substances.  On  absorption  into  the  blood  they  give 
rise  to  headaches,  general  debility  and  in  time,  to  more  serious 
disorders.  Undoubtedly  certain  compounds  of  this  type  are 
produced  normally  by  the  enzymes  of  the  tissue  cells  them- 
selves, and  it  is  probable  that  some  of  them  are  of  importance 
for  the  regulation  of  certain  body  processes.  Adrenaline  is 
such  a  substance. 


PROTEINS  95 

Individual  Groups.    Simple  Proteins 

The  important  characteristics  of  the  proteins  as  a  class  have 
been  considered.  Let  us  now  review  the  properties  of  groups 
and  of  individual  proteins. 

Albumins. — The  albumins  are  found  widely  distributed  in 
nature.  Serum*  albumin  in  the  blood,  ovalbumin  in  egg-white 
and  lactalbumin  in  milk  are  the  best  known  members  of  this 
group.  Compounds  resembling  the  albumins  have  been  found 
in  plants,  although  they  differ  in  some  of  their  properties  from 
the  animal  albumins.  Albumins  contain  no  glycocoU,  and  rela- 
tively much  sulphur  (about  1.6-2.2%).  They  are  soluble  in 
water  and  dilute  salt  solutions,  are  precipitated  by  strong  min- 
eral acids,  by  saturation  with  ammonium  sulphate  in  neutral 
solution  and  by  many  other  precipitants.  They  are  not  pre- 
cipitated by  saturating  a  neutral  solution  with  magnesium  sul- 
phate or  sodium  chloride,  but  if  such  a  solution  is  acidified,  the 
albumins  precipitate.  Albumins  in  solution  coagulate  on  boil- 
ing, if  salts  are  present  and  if  the  solution  is  faintly  acid. 

Globulins. — Globulins  also  are  widely  distributed  in  nature, 
both  in  animals  and  in  plants.  They  often  are  associated  with 
albumins.  Important  members  of  the  group  are  serum  globu- 
lin, fibrinogen  and  its  derivative  fibrin  of  the  blood,  myosinogen 
and  myosin  of  the  muscles,  ovoglobulin  in  eggs,  lactoglobulin  in 
milk,  neuroglobulin  in  nerve  tissue,  and  several  plant  globulins 
such  as  edestin  from  hemp  seed,  legumin  from  peas,  lentils,  etc., 
and  various  others  from  nuts  or  other  materials. 

The  globulins  are  insoluble  in  pure  water,  and  they  may  be 
precipitated  by  pouring  a  solution  of  a  globulin  into  a  large 
volume  of  pure  water,  or  by  dialyzing  out  the  salts  against  dis- 
tilled water  through  a  parchment  membrane.  Globulins  also 
differ  from  albumins  in  containing  glycocoll,  and  in  the  greater 
ease  with  which  they  precipitate  on  the  addition  of  a  neutral 
salt.  Thus  they  are  precipitated  by  half  saturation  with  am- 
monium sulphate  or  by  saturation  with  magnesium  sulphate  or 
sodium  chloride  in  neutral  solution. 


96  PHYSIOLOGICAL    CHEMISTRY 

Fibrinogen  has  the  property  of  clotting  or  coagulating,  and  is 
responsible  for  the  clotting  of  the  blood.  If  freshly  drawn  blood 
is  stirred  or  beaten,  the  fibrinogen  collects  in  stringy  masses 
called  fibrin.  If  washed  free  from  corpuscles  fibrin  is  a  white, 
tough,  elastic  material  which  gives  the  usual  protein  tests. 
Myosinogen  and  myosin  are  the  chief  proteins  of  muscle  tissue. 

Glutelins. — The  glutelins  are  proteins  found*  only  in  the  the 
seeds  of  plants.  The  important  members  of  the  group  are  glu- 
tenin  from  wheat,  oryzenine  from  rice,  and  avenine  from  oats. 
There  probably  are  others.  They  are  characterized  by  being  in- 
soluble in  pure  water,  salt  solutions  or  alcohol.  They  dissolve 
in  dilute  acid  or  alkali. 

Prolamines. — The  prolamines  are  proteins  found  only  in  the 
grains.  They  have  been  obtained  from  all  grains  except  rice. 
The  best  known  members  are  gliadin  from  wheat,  zein  from  corn, 
and  hordcin  from  barley.  The  group  was  named  because  of 
their  high  content  of  proline.  The  prolamines  are  characterized 
by  being  soluble  in  70-80%  alcohol.  They  are  insoluble  in  water. 
They  contain  only  small  amounts  of  arginine  and  histidine  and  no 
lysine. 

Albuminoids. — Albuminoids  are  proteins  obtained  from  a 
wide  variety  of  sources  in  the  animal  world.  Important  mem- 
bers of  the  group  are  keratin  from  horn,  nails,  hair,  hoofs,  etc., 
collagen  from  connective  tissue  and  bone,  its  derivative  gelatine, 
which  properly  does  not  belong  in  this  class  however,  and  elastin, 
from  ligaments  and  connective  tissue.  There  also  are  various 
other  albuminoids.  The  members  of  the  group  are  classed  to- 
gether for  convenience,  since  they  are  insoluble  in  water  and 
most  protein  solvents,  and  are  constituents  of  protective  or  sup- 
porting portions  of  the  body.  Keratin  is  the  chief  constituent 
of  hair,  horn,  nails,  feathers,  the  epidermal  layer  of  the  skin, 
etc.  A  keratin  also  has  been  obtained  from  the  brain  and  nerve 
tissue.  There  probably  are  several  keratins.  The  keratins  con- 
tain much  sulphur  (1-5%).  They  give  the  characteristic  protein 
color  tests. 

Collagen  is  the  chief  constituent  of  the  connective  tissue  and 


PROTEINS  97 

the  chief  organic  constituent  of  bone.  It  occurs  also  in  cartilage. 
There  probably  are  several  collagens.  Collagen,  if  boiled  with 
water  or  dilute  acid,  is  converted  into  gelatine.  Collagen  is  in- 
soluble in  water,  dilute  salt  solutions,  dilute  acids  and  alkalies. 

Gelatine,  obtained  by  boiling  collagen,  swells  up  in  cold  water, 
but  dissolves  in  hot.  If  sufficiently  concentrated,  such  a  solution 
will  set,  on  cooling,  to  a  jelly.  Gelatine  is  not  coagulated  by 
boiling,  nor  will  it  precipitate  on  the  addition  of  strong  mineral 
acids  or  metallic  salts.  It  may  be  precipitated  by  a  variety  of 
reagents,  however,  such  as  alcohol,  tannic  acid,  etc.  Gelatino 
gives  the  biuret  reaction,  but  it  contains  neither  tyrosine  nor 
tryptophane,  and  thus  if  pure  will  not  give  the  Millon  or 
Adamkiewicz  tests.  Gelatine  contains  much  glycocoll.  After 
prolonged  boiling,  a  gelatine  solution  will  no  longer  gel  on  cooling. 

Elastin. — Elastin  occurs  in  connective  tissue,  and  in  largest 
amount  in  the  cervical  ligament.  Fresh  elastin  forms  yellow- 
ish shreds  or  strings  which  are  elastic  in  character.  It  is  in- 
soluble in  water  and  most  of  the  protein  solvents.  Elastin  con- 
tains large  amounts  of  glycocoll  and  leucine.  It  does  not  give 
the  Adamkiewicz  test. 

Histones. — The  histones  are  found  chiefly  in  the  sperma- 
tozoa of  fish,  but  the  globin  portion  of  the  hemoglobin  of  the 
blood  is  usually  classed  in  this  group.  Their  chief  character- 
istic is  a  high  percentage  of  diamino  acids.  In  this  respect  they 
stand  midway  between  the  protamines  and  the  remaining  simple 
proteins.  They  are  basic  in  nature.  The  group  is  not  sharply 
defined. 

Protamines. — The  protamines  are  the  most  basic  of  the  pro- 
teins. They  occur  in  the  ripe  spermatozoa  of  fish.  They  are 
named  from  their  origin,  as  salmine,  from  the  salmon,  sturine 
from  the  sturgeon,  etc.  They  are  characterized  by  their  high 
percentage  of  diamino  acids,  particularly  arginine.  Salmine 
contains  87%  of  arginine.  As  a  result,  solutions  of  protamines 
in  water  are  alkaline  in  reaction.  They  give  the  biuret  test,  but 
most  of  them  do  not  give  the  other  color  tests  for  proteins. 


98  PHYSIOLOGICAL   CHEMISTRY 

They  are  precipitated  fairly  well  by  neutral  salts  and  are  not 
coagulated  by  boiling. 

Conjuguted  Proteins 

The  members  of  this  group  are  made  up  of  protein  combined 
with  some  other  non-protein  substance,  which  is  called  the  pros- 
thetic group.  Among  the  conjugated  proteins  are  substances  of 
great  biological  importance. 

Glycoproteins. — The  glycoproteins  are  compounds  which  on 
breaking  down  yield  a  protein  and  a  relatively  high  percentage 
of  a  carbohydrate  or  carbohydrate  derivative.  Other  proteins 
also  yield  carbohydrates  on  hydrolysis,  but  in  smaller  amounts 
than  the  glycoproteins.  The  group  is  divided  into  two  divisions, 
mucins  and  mucoids.  They  are  very  difficult  to  purify,  espe- 
cially the  mucins,  since  they  are  slimy  in  character,  and  as  a 
result  there  is  much  disagreement  as  to  their  composition  and 
properties.  Mucin  is  secreted  by  the  salivary  glands,  by  certain 
mucous  membranes  and  elsewhere.  The  skin  of  some  of  the 
lower  animals  secretes  large  quantities  of  mucin.  The  mucins  are 
distinguished  from  the  mucoids  by  their  slimy  character  and  by 
the  fact  that  on  precipitation  with  acetic  acid,  they  do  not  re- 
dissolve  in  excess  of  acid.  The  mucoids  are  found  in  tendons,  in 
cartilage,  in  the  cornea  and  crystalline  lens,  in  egg  white  and 
various  other  places.  Some  authors  divide  this  group  again  into 
mucoids  and  chondroproteins.  The  mucoid  of  tendon  or  carti- 
lage may  be  extracted  with  lime  water.  On  acidifying  with 
acetic  acid,  the  mucoid  precipitates,  but  it  will  redissolve  in  ex- 
cess. Both  the  mucins  and  the  mucoids  contain  a  relatively  high 
amount  of  sulphur,  a  part  of  which,  at  least,  is  in  the  form  of 
oxidized  sulphur.  On  hydrolysis  the  mucoids  yield  a  substance 
chondroitic  acid  or  chondroitin  sulphuric  acid,  which  has  been 
the  subject  of  much  study.  The  carbohydrate  radicle  in  the 
glycoproteins  is  usually  glucosamine  or  galactosamine. 

Phosphoproteins.— The  phosphoproteins/  are  compoun;ds  of 
importance  because  they  furnish  a  large  share  of  the  protein 


PROTEINS  99 

nourishment  of  the  growing  young  of  animals  and  birds.  There 
are  two  important  members  of  the  group,  the  casein  of  milk  and 
the  vitellin  of  egg  yolk.  They  are  characterized  by  containing  a 
relatively  high  amount  of  phosphorus  (about  1%)  which  is 
present  in  some  form  neither  lecithin  nor  nucleic  acid.  On 
digestion,  the  phosphoproteins  leave  a  difficultly  digestible  resi- 
due known  as  pseudonuclein.  The  phosphoproteins  are  acid  in 
character.  In  their  solubilities  and  precipitation  properties  they 
resemble  both  the  globulins  and  the  nucleoproteins.  They  may 
be  distinguished  from  the  former  by  their  phosphorus  content, 
and  from  the  latter  by  the  fact  that  they  yield  no  purine  bases 
on  hydrolysis.  Cow's  milk  contains  about  3-4%  casein,  human 
milk  about  0.5-1.5%.  Casein  is  not  coagulated  by  boiling.  It  is 
insoluble  in  water,  but  dissolves  readily  in  dilute  alkalies.  From 
such  a  solution,  and  from  milk,  casein  is  precipitated  by  the 
addition  of  a  small  amount  of  acid.  This  occurs  also  in  the 
souring  of  milk.  Bacteria  decompose  the  lactose,  forming  or- 
ganic acids.  From  this  acid  solution  the  casein  is  precipitated, 
causing  the  characteristic  clotting  of  sour  milk.  It  may  be  pre- 
pared by  diluting  milk,  and  adding  a  small  amount  of  acetic 
acid.  To  free  the  casein  from  fat  it  may  be  redissolved  in  sodium 
carbonate  and  reprecipitated  with  acid.  This  process  should 
be  repeated  several  times  if  a  pure  product  is  desired.  Casein 
contains  no  glycocoll,  but  little  cystine  and  relatively  much  tryp- 
tophane. On  hydrolysis  it  yields  also  several  acids  which  are 
not  obtained  from  other  proteins.  Casein  is  clotted  by  a  fer- 
ment, rennin,  which  occurs  in  digesive  juices,  particularly  the 
gastric  juice.  It  may  be  well  to  state  in  this  connection  that  the 
nomenclature  of  casein  and  its  allied  substances  is  somewhat 
confused.  By  many  investigators  the  substance  as  it  occurs  in 
milk  is  called  easeinogen.  This  is  converted  by  rennin  into 
another  substance,  paracasein.  Paracasein  differs  from  easein- 
ogen at  least  in  one  respect,  the  insolubility  of  its  calcium  salt. 
The  calcium  salt  of  easeinogen  is  soluble,  that  of  paracasein  in- 
soluble. Since  calcium  salts  are  found  normally  in  milk,  para- 
casein is  precipitated  as  the  familiar  curdy  material.    If  calcium 


100  PHYSIOLOGICAL    CHEMISTRY 

is  previously  removed  by  adding  an  oxalate  to  the  milk,  the  para- 
casein is  formed  from  caseinogen  by  rennin,  but  will  not  precip- 
itate. Subsequent  addition  of  excess  of  a  soluble  calcium  salt 
will  cause  precipitation  even  if  the  milk  has  been  boiled  to  de- 
stroy the  rennin.  In  the  transformation  of  caseinogen  into  para- 
casein a  protein  like  substance  called  albumose,  or  ''whey  pro- 
tein" is  split  off.  This  clotting  is  the  first  step  in  the  digestion 
of  casein  in  the  stomach,  and  serves  to  prevent  the  milk  from 
passing  on  too  quickly  into  the  intestine. 

VitelUn. — Vitellin  is  found  in  egg  yolk  and  may  be  prepared 
by  extracting  the  yolk  with  ether  to  remove  lecithin,  cholesterol, 
etc.  The  residue  is  dissolved  in  salt  solution  and  reprecipitated 
by  pouring  into  a  large  volume  of  water.  To  remove  the  last  of 
the  lecithin  it  is  necessary  to  extract  with  boiling  alcohol. 

Hemoglobins.— This  group  often  is  called  the  group  of  chro- 
moproteins,  since  its  members  usually  are  colored  substances. 
The  most  important  of  them,  is  hemoglobin  or  oxyhemoglobin, 
the  red  coloring  matter  of  the  blood.  This  compound  is  con- 
tained in  the  red  corpuscles  in  higher  animals.  In  some  of  the 
lower  animals  it  is  simply  dissolved  in  the  blood  plasma.  It,  or  a 
substance  closely  allied  to  it,  occurs  also  in  red  muscle.  The  hemo- 
globin fulfils  an  important  function  in  the  body,  that  of  trans- 
porting oxygen  from  the  lungs  to  the  tissues.  If  hemoglobin  is 
exposed  to  a  plentiful  supply  of  oxygen,  as  is  the  case  when  the 
red  corpuscles  are  passing  through  the  capillaries  surrounding 
the  tiny  air  spaces,  the  alveolae  in  the  lungs,  it  takes  up  oxygen 
and  is  converted  into  oxyhemoglobin.  When  the  corpuscles  con- 
taining this  oxyhemoglobin  reach  the  tissues,  again  they  pass 
through  a  fine  network  of  capillaries.  Here,  however,  the  oxygen 
supply  is  very  low,  as  oxygen  is  rapidly  used  up  by  the  cells  in 
oxidation  processes.  The  oxyhemoglobin  gives  up  its  oxygen, 
becoming  hemoglobin  again,  and  the  oxygen  passes  out  through 
the  capillary  walls  to  the  region  of  low  oxygen  supply.  Re- 
turning to  the  lungs,  the  hemoglobin  takes  up  a  fresh  supply  of 
oxygen,  and  again  carries  it  to  the  tissues.  This  property  of 
hemoglobin  may  be  demonstrated  easily  as  follows:    On  adding 


PROTEINS  101 

an  equal  volume  of  water  to  the  blood,  the  hemoglobin  diffuses 
out  of  the  corpuscles  and  the  solution  becomes  clear.  The  blood 
is  said  to  be  ''laked."  If  the  mouth  of  the  test  tube  is  closed 
with  the  thumb  or  a  stopper  and  the  liquid  shaken  well,  the 
color  of  the  solution  becomes  a  brilliant  red.  It  now  contains 
oxyhemoglobin,  the  color  of  which  is  a  much  brighter  red  than 
that  of  hemoglobin,  this  substance  gives  arterial  blood  its  bright 
red  color.  If  a  mild  reducing  agent  is  added  to  this  oxyhemoglo- 
bin the  color  becomes  much  darker.  The  oxygen  has  been  taken 
away  from  the  oxyhemoglobin,  which  has  now  become  hemo- 
globin. This  substance  gives  venous  blood  its  dark  red  color. 
This  process  may  be  repeated  indefinitely.  A  convenient  mild 
reducing  agent  is  Stokes'  fluid  which  contains  2%  ferrous  sul- 
phate, 3%  tartaric  acid  and  ammonia  sufficient  to  redissolve  the 
precipitate  which  first  forms  on  adding  this  reagent.  The  amount 
of  oxygen  which  can  be  carried  by  a  given  amount  of  blood  varies 
somewhat.  On  the  average  from  100  c.c.  of  fully  oxygenated 
blood  19-20  c.c.  of  oxygen  can  be  obtained  (measured  at  0°  C.  and 
760  mm  of  mercury  pressure) .  A  portion  of  this  is  simply  dis- 
solved in  the  blood  plasma,  but  most  of  it  is  combined  with 
hemoglobin.  The  tissues  do  not  remove  all  of  the  oxygen  from 
the  blood,  for  venous  blood  still  contains  considerable  oxyhemo- 
globin. From  100  c.c.  of  venous  blood  an  average  of  15  c.c.  of 
oxygen  may  be  obtained,  although  this  value  varies  considerably. 
The  molecular  weight  of  hemoglobin  is  very  large,  probably 
about  16,000,  and  one  molecule  of  hemoglobin  is  believed  to  com- 
bine with  one  molecule  of  oxygen  (Og).  The  air  normally  con- 
tains about  20%  oxygen.  This  percentage  may  be  much  lowered, 
however,  before  the  amount  of  oxyhemoglobin  in  a  hemoglobin- 
oxyhemoglobin  mixture  will  be  greatly  diminished.  If  the  per 
cent  of  oxygen  is  reduced  to  13%  of  an  atmosphere  the  mixture 
will  still  contain  93%  oxyhemoglobin  and  only  7%  hemoglobin. 
Hemoglobin  may  be  split  into  two  parts,  globin,  a  protein, 
perhaps  a  histone,  and  hemoehromogen.  From  oxyhemoglobin 
hematin  is  obtained  in  place  of  hemoehromogen.  Grlobin  makes 
up  about  9'5%  of  the  hemoglobin,  hemoehromogen  about  4%. 


102  PHYSIOLOGICAL    CHEMISTRY 

The  corpuscles  contain  about  30%  of  hemoglobin,  an  amount 
much  greater  than  could  be  dissolved  in  an  equal  amount  of 
fluid,  so  it  must  be  assumed  that  hemoglobin  is  held  in  some 
loose  combination  with  the  other  constituents  of  the  corpuscles. 
Hemoglobin  and  many  of  its  derivatives  contain  iron  to  the 
extent  of  about  3%.  This  iron  seems  to  be  directly  connected 
with  the  ability  of  hemoglobin  to  combine  with  oxygen. 

Oxyhemoglobin  may  be  crystallized  with  comparative  ease. 
Oxyhemoglobins  from  different  animals  form  crystals  of  greatly 
varying  form,  and  may  be  crystallized  with  varying  degrees  of 
ease.  Those  easiest  to  crystallize  are  oxyhemoglobin  of  the 
guinea  pig,  which  forms  tetrahedra,  of  the  rat  (rhomboids),  of 
the  squirrel  (hexagons),  of  the  horse,  dog,  etc.  Oxyhemoglobin 
from  man  (long  rods,  rhomboids),  ox,  and  other  animals  may  be 
obtained  in  crystalline  form,  but  with  greater  difficulty.  Crys- 
tals of  the  types  which  crystallize  easily  may  be  obtained  by  a 
variety  of  methods.  That  of  Reiehert  consists  in  adding  1-5%  of 
solid  ammonium. oxalate  to  blood,  either  before  or  after  laking 
or  defibrinating.  A  drop  of  this  blood  placed  on  a  slide  will 
crystallize  quickly.  On  standing  in  the  ice  chest  very  beautiful 
large  crystals  form. 

Reiehert  and  Brown  have  studied  the  oxyhemoglobin  crystals 
from  the  blood  of  a  great  many  animals  and  maintain  that  bio- 
logical relationships  may  be  traced  in  the  crystal  form,  since 
oxyhemoglobins  from  animals  of  the  same  species  crystalize  in 
similar  forms.  Hemoglobin  is  more  soluble  than  oxyhemoglobin, 
and  is  thus  much  more  difficult  to  crystallize. 

Detection  of  Hemoglobin. — In  medical  practice,  and  also  in 
medico-legal  cases  it  often  is  of  the  greatest  importance  to  de- 
tect and  identify  hemoglobin  or  its  derivatives.  This  is  done  in 
a  variety  of  ways  as  follows. 

Catalytic  Activity  of  Blood. — If  hydrogen  peroxide  is  added 
to  blood,  or  even  to  blood  diluted  to  the  point  where  the  solution 
is  no  longer  colored,  the  peroxide  is  decomposed,  and  bubbles  of 
oxygen  gas  are  given  off.    If  the  blood  is  first  boiled,  no  action 


PROTEINS  103 

takes  place.    Thus  it  appears  that  the  blood  contains  an  enzyme 
capable  of  decomposing  peroxides. 

Hemin  Test. — One  of  the  best  tests  for  blood  is  the  prepara- 
tion of  hemin  crystals.  Hemin  is  the  hydrochloride  of  hema- 
tin.  The  hemin  test  will  not  differentiate  between  the  blood  of 
man  and  other  animals.  Although  hemoglobins  from  different 
animals  differ,  it  is  the  globin  portion  which  varies,  and  the 
hematin  from  different  animals  is  undoubtedly  the  same.  The 
hemin  test  is  verj'^  delicate.  The  suspected  stains  are  extracted 
with  water  or  weak  alkali,  the  solution  evaporated  to  dryness 
and  the  residue  used  for  the  test.  If  the  solution  is  very  dilute, 
the  pigment  may  be  precipitated  with  tannic  acid,  and  the  test 
made  on  the  precipitate. 

The  test  is  performed  by  evaporating  a  drop  of  blood  or  sus- 
pected material  to  dryness  on  a  slide,  adding  sodium  chloride  and 
a  few  drops  of  glacial  acetic  acid.  The  mixture  is  covered  with  a 
cover  glass  and  heated  carefully  until  the  acid  boils.  On  cool- 
ing, brown  or  reddish  brown  rhomboidal  crystals  of  hemin  form. 
If  crystals  do  not  form  the  first  time,  the  mixture  should  be 
boiled  again  as  above.  It  may  be  necessary  to  repeat  the  process 
several  times.  As  hematin  is  very  stable,  blood  stains  which 
have  putrefied,  or  which  are  even  centuries  old  still  will  give 
the  test.  Wood  smoke  and  swamp  water  do  not  destroy  hem- 
atin; it  may  be  destroyed  if  certain  moulds  have  grown  on  the 
stain  however.  It  is  of  interest  that  the  excreta  spots  of  blood 
sucking  insects  will  give  a  positive  hemin  test. 

Guaiac  Test. — Benzidine  Test. — Blood  has  the  property  of 
transferring  oxygen  from  hydrogen  peroxide  to  easily  oxidized 
chemical  substances  such  as  guaiac,  benzidine  and  a  variety  of 
other  compounds.  On  adding  a  small  amount  of  a  freshly  pre- 
pared 1  %  alcoholic  gum  guaicum  solution  to  a  solution  con- 
taining blood,  and  then  a  small  volume  of  3%  hydrogen  peroxide, 
a  bluish  green  color  develops.  Old  turpentine  which  has  not 
recently  stood  in  direct  sunlight  may  be  used  in  place  of  the 
hydrogen  peroxide.  In  performing  the  benzidine  test  it  is  best 
to  dissolve  in  glacial  acetic  acid  the  amount  of  benzidine  which 


104  PHYSIOLOGICAL    CHEMISTRY 

can  be  taken  on  a  knife  point,  then  add  an  equal  volume  of 
hydrogen  peroxide  and  the  liquid  to  be  tested.  If  blood  is  pres- 
ent, the  color  produced  will  be  a  greenish  blue.  If  blood  has 
been  diluted  200,000  times  it  still  will  give  a  very  definite  posi- 
tive reaction.  Other  substances  also  respond  to  these  tests,  such 
as  milk,  living  matter,  etc.  A  slice  of  raw  carrot  gives  a  very 
good  test.  Milk,  and  living  matter,  (carrot)  if  boiled,  no  longer 
will  give  the  test,  whereas  blood  gives  it  quite  as  readily  after 
boiling.  Evidently  in  the  case  of  blood  the  material  responsible 
for  the  test  is  not  an  enzyme.  As  negative  tests,  these  color 
tests  may  be  taken  to  indicate  the  absence  of  blood.  As  positive 
tests,  however,  they  are  not  conclusive  without  further  con- 
firmation from  other  tests.  Also  they  do  not  distinguish  human 
blood  from  that  of  other  animals. 

Absorption  Spectra  of  Oxyhemoglobin  and  Hemoglobin.— 
Hemoglobin  and  many  of  its  derivatives  show  characteristic  ab- 
sorption spectra.  Thus  a  spectroscopic  investigation  often  is 
of  the  greatest  value  in  detecting  blood  in  feces,  urine,  gastric 
contents  or  in  stains  in  medico-legal  work.  The  student  is  re- 
ferred to  the  discussion  of  absorption  spectra  under  pentoses 
in  the  chapter  on  carbohydrates.  The  nature  of  the  absorption 
bands  depends  not  only  upon  the  substance  present,  but  the  con- 
centration of  the  solution  and  thickness  of  the  layer  through 
which  the  light  passes.  Blood  diluted  ten  times  with  water,  and 
observed  with  a  spectroscope  in  a  flat  sided  cell  about  one  centi- 
meter in  thickness  allows  only  a  little  red  light  to  pass  through. 
If  the  solution  is  diluted  however,  it  shows  two  absorption  bands 
near  together  between  the  D  and  E  Frauenhofer  lines.  On  fur- 
ther dilution  these  lines  become  fainter  and  at  sufficient  dilution, 
finally  disappear. 

If  Stokes '  reagent  is  added  to  an  oxyhemoglobin  solution,  thus 
converting  the  oxyhemoglobin  into  hemoglobin  the  double  bands 
give  place  to  a  single  continuous  band  in  about  the  same  loca- 
tion. 

The  amount  of  hemoglobin  or  oxyhemoglobin  in  blood  or 
other  fluids  may  be  estimated  by  various  means,  the  most  con- 


PROTEINS  105 

vienient  consisting  in  comparing  blood  or  diluted  blood  with  a 
scale  giving  reds  of  different  intensities  corresponding  to  definite 
concentrations  of  hemoglobin. 

Derivatives  of  the  Hemoglobins. — Carbon  Monoxide-Hemo- 
glohin. — Hemoglobin  forms  compounds  with  various  gases  other 
than  oxygen  such  as  carbon  monoxide,  carbon  dioxide,  etc.  Car- 
bon monoxide,  which  is  a  constituent  of  illuminating  gas  com- 
bines with  hemoglobin  in  molecular  proportions.  Its  union  ap- 
parently is  firmer  than  that  of  oxygen,  and  apparently  it  com- 
bines with  hemoglobin  at  the  same  place  as  does  oxygen,  for  if 
both  gases  are  present,  carbon  monoxide  hemoglobin  is  formed, 
and  the  taking  up  of  oxygen  is  interfered  with.  The  carbon 
monoxide  may  be  removed,  however,  by  passing  a  stream  of 
air  through  the  liquid  for  some  time,  and  oxyhemoglobin  will  be 
formed.  In  cases  of  asphyxiation  by  illuminating  gas,  carbon 
monoxide  is  found  in  the  blood,  thus  preventing  the  proper 
transportation  of  oxygen  to  the  tissues. 

Solutions  of  carbon  monoxide  hemoglobin  are  a  bright  cherry 
red.  The  absorption  bands  look  much  like  those  of  oxyhemo- 
globin,— two  dark  bands  between  the  D  and  the  E  lines.  They 
are  slightly  nearer  the  violet  end  of  the  spectrum  however,  and 
on  adding  Stokes'  reagent  to  the  mixture  they  do  not,  as  does 
the  hemoglobin  spectrum,  give  place  to  a  single  broad  band. 

Carbon  dioxide  apparently  combines  with  hemoglobin  at  a 
point  different  from  that  at  which  oxygen  combines,  as  combin- 
ing with  one  does  not  prevent  hemoglobin  from  combining  with 
the  other  also. 

MetJiemoglohin. — ^Methemoglobin  is  a  compound  derived  from 
oxyhemoglobin.  It  contains  the  same  amount  of  oxygen  as  oxy- 
hemoglobin, but  the  oxygen  is  more  firmly  united  than  in  oxy- 
hemoglobin. Methemoglobin  is  found  in  the  blood  after  poison- 
ing Avith  chlorates,  amyl  nitrite,  etc.,  and  is  found  occasionally 
in  the  urine,  in  transudates,  cystic  fluids,  and  elsewhere.  Out- 
side the  body  it  may  be  prepared  for  study  by  adding  fresh 
potassium  ferricyanide  solution,  permanganate  or  other  sub- 
stances to  oxyhemoglobin  solutions.    The  solution  turns  a  muddy 


106  PHYSIOLOGICAL   CHEMISTRY 

brown.  On  dilution  and  observation  with  the  spectroscope,  a 
dark  absorption  band  between  the  C  and  D  lines  is  observed.  Two 
fainter  bands  in  the  position  of  the  oxyhemoglobin  bands  are 
considered  by  some  investigators  to  be  due  to  the  presence  of  a 
small  amount  of  this  latter  pigment.  On  adding  Stokes'  reagent 
to  a  methemoglobin  solution,  the  substance  is  changed  first  into 
oxyhemoglobin,  and  this  into  hemoglobin,  with  corresponding 
changes  in  the  spectrum. 

On  adding  an  alkali  to  a  methemoglobin  solution,  alkaline 
methemoglobin  is  formed,  which  gives  a  characteristic  spectrum 
of  its  own. 

Acid  Hematin. — Hematin  is  the  compound  into  which  oxy- 
hemoglobin may  be  split  by  the  action  of  acids  or  other  agents. 
It  contains  the  iron  of  the  hemoglobin  molecule,  and  is  com- 
paratively simple  in  structure.  It  has  been  shown  to  contain 
four  pyrrol  rings. 

HC  — CH 

HO       CH 

\/ 

N 

H 

Pyrrol 

Hematin  is  formed  from  oxyhemoglobin  by  the  action  of  gas- 
tric and  pancreatic  juices.  It  is  found  thus  in  the  feces  after 
gastric  or  intestinal  hemorrhage,  but  also,  of  course,  after  a 
meal  of  bloody  meat.  Hematin  gives  the  brown  color  to  cooked 
meat.  It  may  be  prepared  by  adding  blood  to  glacial  acetic  acid 
and  ether.  The  absorption  spectrum  of  acid  hematin  is  variable 
with  the  kind  of  acid  used  in  its  preparation  and  other  condi- 
tions. If  prepared  as  above,  however,  it  shows  a  sharp  dark  band 
between  the  C  and  D  lines  and  a  fainter  broad  band  between  the 
D  and  F  lines.     Alkaline  hematin  shows  characteristic  bands. 

H emocliromogen. — Hemochromogen,  or  reduced  alkaline  hem- 
atin is  obtained  by  splitting  off  the  globin  from  hemoglobin  by 
the  action  of  acids  or  alkalies,  or  by  the  reduction  of  alkaline 
hematin  with  Stokes'  reagent.     Like  hematin,  it  also  contains 


PROTEINS  107 

iron.  Oxygen  forms  with  hemochromogen  a  more  stable  com- 
pound than  with  hemoglobin.  Hemochromogen  shows  character- 
istic absorption  bands. 

H omotoporphyrin. — Hemotoporphyrin  is  an  iron  free  deriva- 
tive of  hemoglobin  which  may  be  prepared  by  adding  blood  to 
concentrated  sulphuric  acid.  The  liquid  becomes  purple  and  may 
be  shown  to  contain  hemotoporphyrin.  It  gives  two  absorption 
bands,  one  on  each  side  of  the  D  line.  If  the  solution  is  made 
alkaline,  the  acid  hemotoporphyrin  is  changed  to  alkaline  hemo- 
toporphyrin, which  shows  a  different  spectrum.  Hemotopor- 
phyrin is  found  in  the  urine  in  considerable  amounts  after  sul- 
fonal  poisoning  and  the  statement  has  been  made  that  small 
amounts  occur  in  normal  urine.  Hemotoporphyrin  is  interesting 
from  the  fact  that  it  is  closely  related  to  phylloporphyrin,  a  de- 
rivative of  chlorophyl,  the  green  coloring  matter  in  plants.  In 
this  same  connection  it  is  also  interesting  that  hemotoporphyrin 
acts  as  a  photo-sensitizer,  making  animals  sensitive  to  light.  If 
hemotoporphyrin  is  injected  into  a  white  mouse,  no  ill  effects  ap- 
pear so  long  as  the  animal  is  kept  in  the  dark.  If  exposed  to 
strong  sunlight,  however,  it  will  develop  dyspnoea,  swelling  of 
the  ears,  oedema  of  the  skin,  and  ultimately  will  die.  In  man,  in 
some  skin  diseases  connected  with  the  action  of  strong  sunlight, 
hemotoporphyrin  has  been  observed  in  the  urine. 

Fate  of  blood  pigment  in  the  body.  Hemoglobin  from  broken 
down  corpuscles  is  converted  into  bile  pigments,  and  thus  is  the 
source  of  these  coloring  matters.  The  transformation  in  mam- 
mals is  made  in  the  liver  itself  and  undoubtedly  to  a  considerable 
extent  in  the  spleen,  although  this  latter  organ  probably  carries 
out  only  the  first  stages  of  the  process.  The  bile  pigments,  bili- 
rubin and  biliverdin,  and  also  urobilin,  a  yellow  pigment  in  the 
urine  thus  come  from  the  broken  down  hemoglobin  of  the  blood. 

Nucleoproteins. — The  nucleoproteins  are  found  in  the  nuclei 
of  all  cells,  both  plant  and  animal.  It  has  been  suggested  that 
they  occur  in  other  portions  of  the  cell,  or  in  the  blood  plasma, 
but  this  is  doubtful,  although  their  decomposition  products  may 
occur  there.    Since  the  cell  nucleus  is  intimately  concerned  in  the 


108  PHYSIOLOGICAL    CHEMISTRY 

process  of  cell  division,  and  thus  in  reproduction  and  growth, 
any  substances  found  regularly  and  characteristically  in  the 
nucleus  will  be  of  great  interest.  The  nucleoproteins  are  char- 
acterized by  containing  nucleic  acid,  which  may  be  obtained  from 
them  by  splitting  off  the  protein  portion  of  the  molecule.  This 
process  can  be  carried  on  in  two  stages,  part  of  the  protein  split- 
ting off  more  easily,  leaving  a  product  called  nuclein,  as  in  the 
action  of  gastric  juice  on  a  nucleoprotein.  By  the  action  of 
caustic  alkali  the  remaining  protein  in  nuclein  may  be  split  off, 
leaving  nucleic  acid. 

This  nucleic  acid  is  by  far  the  more  interesting  part  of  the 
nucleoprotein  molecule,  and  it  has  been  much  studied.  By 
vigorous  hydrolysis  nucleic  acid  may  be  split  into  four  kinds  of 
material  (1)  purine  bases,  (2)  pyrimidine  bases,  (3)  a  carbohy- 
drate, and  (4)  phosphoric  acid.  Four  purine  bases  are  obtained, 
adenine,  guanine,  hypoxanthine  and  xanthine.  Only  the  first 
two  are  considered  to  be  present  in  the  original  molecule,  the 
last  two  being  formed  from  the  others  in  the  process  of  hydro- 
lysis. In  the  body  a  fifth  member  of  the  group  is  formed  from 
the  others,— uric  acid. 

HN  —  C  =  0 

I        I 
0  =  C       C  —  NH 

\ 

c  =  o 

/ 

NH  —  C  —  NH 

Uric  Acid. 

The  pyrimidine  bases  are  somewhat  closely  allied  to  the 
purine  bases.  Uracil,  thymine  and  cytosine  are  the  three  mem- 
bers of  the  group  obtained  from  nucleic  acid. 

The  carbohydrate  portion  of  the  nucleic  acid  is  sometimes  a 
pentose  and  sometimes  a  hexose  or  allied  substance. 

The  fourth  product  is  phosphoric  acid,  H3PO4. 

The  structure  of  the  nucleic  acids  has  been  worked  out,  and 
the  student  is  referred  to  larger  works  for  further  discussion  of 
this  subject. 


PROTEINS  109 

The  nucleoproteins  may  be  extracted  from  tissue,  such  as  the 
pancreas  by  boiling  with  water.  On  acidifying  with  acetic  acid 
the  nucleoprotein  will  precipitate.  In  this  and  other  properties 
the  nucleoproteins  resemble  the  globulins  and  the  phospho- 
proteins.  From  both  of  these  groups  they  may  be  distinguished 
by  their  content  of  purine  bases.  The  nucleoproteins  contain 
about  the  same  per  cent  of  phosphorus  as  the  phosphoproteins. 

Lecithoproteins. — The  lecithoproteins  are  compounds  made  up 
of  protein  combined  with  lecithin  or  some  other  phosphatid. 
Little  definite  information  is  available  concerning  these  com- 
pounds. Some  investigators  put  vitellin  in  this  class  instead  of 
among  the  phosphoproteins,  for  vitellin  can  only  be  freed  from 
the  lecithin  which  occurs  with  it,  by  extraction  with  hot  alcohol. 
It  has  been  suggested  also  that  the  residue  of  the  blood  corpuscles 
after  removing  hemoglobin  contains  a  lecithoprotein.  Our  in- 
formation about  the  group  is  still  in  a  fragmentary  and  unsatis- 
factory state. 

Derived  Proteins 

The  derived  proteins  are  produced  when  proteins  are  acted  on 
by  various  agents.  The  group  is  divided  into  primary  and  sec- 
ondary protein  derivatives.  In  making  the  former  the  proteins 
are  not  greatly  changed  in  their  properties.  In  making  the  sec- 
ondary derivatives,  extensive  alteration  or  breaking  down  occurs. 
The  primary  derivatives  are  again  divided  into  proteans,  meta- 
proteins,  and  coagulated  proteins.  The  secondary  derivatives  are 
subdivided  into  proteoses,  peptones,  and  peptids. 

Primary  Protein  Derivitives. — Proteans. — Proteans  probably 
may  be  formed  from  most  of  the  simple  proteins.  Those  formed 
from  the  globulins  have  been  most  studied.  If  edestin,  the  glo- 
bulin from  hemp  seed,  is  dissolved  in  the  smallest  possible 
amount  of  hydrochloric  acid,  it  may  be  precipitated  by  adding 
a  small  amount  of  sodium  chloride.  On  adding  stronger  salt 
solution  a  portion  of  this  precipitate  will  not  dissolve.  As  often 
as  the  above  process  is  repeated,  a  further  portion  of  the  protein 
becomes  insoluble.    The  same  results  are  obtained  by  the  action 


110  PHYSIOLOGICAL    CHEMISTRY 

of  weak  alkalies,  or  the  initial  action  of  enzymes  on  proteins. 
The  resulting  substances  are  called  proteans,  and  are  named 
from  the  material  from  which  they  are  prepared, — thus  edestan 
from  edestin,  myosan  from  myosin,  etc.  They  differ  very  slightly 
from  unchanged  proteins,  and  undoubtedly  only  minor  rear- 
rangements in  the  protein  molecule  have  occurred  in  their 
formation. 

Metaproteins. — Metaproteins  are  formed  from  the  proteins 
by  the  action  of  alkalies  or  acids,  either  dilute  or  concentrated. 
They  are  sometimes  called  albuminates.  If  a  very  dilute  acid 
or  alkali  is  allowed  to  act  on  a  protein  at  body  temperature  for 
some  time,  perhaps  a  half  hour,  it  is  quite  evident  that  some 
change  has  taken  place  in  the  protein  for  if  the  faintly  acid 
solution  of  the  substance  is  boiled,  no  coagulation  takes  place. 
Both  acid  and  alkali  metaprotein,  formed  by  using  dilute  acid 
or  alkali,  wall  dissolve  in  a  slight  excess  either  of  acid  or  of 
alkali.  If  the  solution  is  made  exactly  neutral,  however,  the 
metaproteins  will  precipitate.  If  this  precipitate  is  suspended 
in  water  and  boiled,  it  is  coagulated,  and  no  longer  can  be  dis- 
solved by  adding  dilute  alkali  or  acid.  Dissolving  alkali  meta- 
protein in  acid,  or  acid  metaprotein  in  alkali  does  not  change 
these  substances  into  one  another.  Perhaps  acid  metaprotein 
may  be  changed  into  alkali  metaprotein  by  longer  action  of  the 
alkali,  but  the  reverse  process  surely  does  not  take  place.  Alkali 
metaprotein  is  formed  more  quickly  than  is  acid  metaprotein, 
and  in  the  former  process  some  nitrogen  and  sulphur  are  split 
off.  Acid  metaprotein  is  particularly  interesting  because  it  is 
formed  as  the  first  stage  in  the  digestion  of  most  proteins  in  the 
stomach.  Gastric  juice  contains  hydrochloric  acid  which  forms 
acid  metaprotein  from  the  proteins  of  the  food,  thus  bringing 
them  into  solution.  The  metaproteins  behave  in  most  respects 
like  proteins,  and  apparently  the  protein  molecule  is  not  ex- 
tensively altered  when  they  are  formed. 

Coagulated  Proteins. — Coagulated  proteins  are  formed  when 
acid  solutions  of  most  proteins  are  heated  to  high  tempera- 
tures, when  most  proteins  are  allowed  to  stand  under  alcohol,  or 


PROTEINS  111 

are  acted  on  by  certain  enzymes,  and  perhaps  by  other  means. 
They  are  characterized  by  their  insolubility  in  the  usual  mild 
protein  solvents,  such  as  water,  salt  solutions,  etc.  Little  is 
known  of  what  takes  place  in  the  protein  molecule  in  coagula- 
tion. Although  coagulated  proteins  are  fairly  insoluble,  still 
they  will  dissolve  in  strong  acids  or  alkalies,  or  in  dilute  acids 
or  alkalies  on  warming  slightly  for  some  time.  In  the  latter 
case  they  are  converted  into  metaproteins.  In  the  process  of 
cooking  food,  most  of  the  proteins  are  coagulated,  but  they  are 
converted  into  metaprotein  by  the  acid  of  the  gastric  juice,  and 
then  digested.  Some  proteins  are  digested  more  easily  after 
coagulation,  others  less  so.  Coagulated  proteins  give  all 
color  tests  given  by  the  original  protein.  The  protein  mole- 
cule evidently  has  not  been  greatly  altered  or  broken  down  in 
their  formation. 

Secondary  Protein  Derivatives. — Proteoses  and  Peptones 
are  formed  from  proteins  by  the  action  of  strong  acids  or  alka- 
lies, or  by  the  action  of  enzymes.  The  protein  molecule  is  broken 
up  into  fragments  of  varying  size  and  properties.  From  what 
is  known  of  the  size  and  complexity  of  the  protein  molecule  it 
is  easy  to  understand  the  great  diversity  in  size,  composition  and 
properties  of  the  products  formed  on  splitting  it  into  fragments. 
Much  labor  has  been  spent  in  the  study  of  these  compounds.  As 
yet  little  is  known  of  the  individual  substances.  For  conven- 
ience, they  are  divided  according  to  the  concentration  of  am- 
monium sulphate  required  to  precipitate  them.  Those  which 
precipitate  on  saturating  with  ammonium  sulphate  are  called 
proteoses.  Those  which  will  not  precipitate  on  saturating  with 
ammonium  sulphate  are  called  peptones.  Such  a  method  of 
separation  is  unsatisfactory,  but  it  serves  its  purpose  until  more 
is  known  of  the  constitution  of  the  individual  substances.  It  is 
now  known  that  factors  other  than  the  size  of  the  molecule  affect 
the  ease  with  which  a  substance  may  be  precipitated  with  am- 
monium sulphate.  Thus  substances  of  the  above  nature  which 
contain  tyrosin,  cystin  and  tryptophane  are  precipitated  at 
lower  concentrations  of  ammonium  sulphate  than  are  those  even 


112  PHYSIOLOGICAL   CHEMISTRY 

of  larger  molecular  weight  in  which  these  amino  acids  are  miss- 
ing. A  study  of  the  proteoses  and  peptones  has  developed  the 
important  fact  that  the  breaking  down  of  the  protein  molecule 
is  by  no  means  a  symmetrical  process.  In  fact,  it  probably  con- 
sists in  breaking  off  fragments  of  the  greatest  diversity,  includ- 
ing some  of  the  amino  acids  themselves  even  in  the  early  stages 
of  the  process.  There  appear  to  be  certain  combinations  or 
groups, — "nuclei"  we  may  call  them,  in  the  protein  molecule 
which  are  quite  resistent  to  hydrolyzing  agents,  and  even  after 
very  vigorous  hydrolysis  such  groups  may  remain  intact. 
Although  the  proteoses  and  peptones  are  simpler  in  structure 
than  the  proteins,  they  still  are  quite  complex  substances  and 
contain  many  amino  acid  molecules,  many  more  than  the  poly- 
peptid  of  18  amino  acids  built  up  by  Fischer, 

By  boiling  with  strong  acids  or  alkalies,  or  by  the  action  of 
certain  enzymes,  proteoses  and  peptones  are  split  into  amino 
acids  as  might  be  expected. 

Neither  proteoses  nor  peptones  coagulate  on  boiling.  Both 
groups  give  the  biuret  test.  The  color  is  redder  than  that  given 
by  proteins,  however.  They  usually  give  the  xanthoproteic  test. 
Their  response  to  the  Millon's  and  Adamkeiwicz  reactions  de- 
pends on  the  presence  of  tyrosine  and  tryptophane  in  the  par- 
ticular proteose  or  peptone  under  investigation.  Proteoses  usu- 
ally give  these  tests.  Peptones  seldom  do.  Concentrated  nitric 
acid  precipitates  some  proteoses.  These  are  called  primary  pro- 
teoses. It  does  not  precipitate  "secondary  proteoses"  or  pep- 
tones. Peptones  will  diffuse  fairly  well  through  an  animal  mem- 
brane. 

Peptids  are  substances  consisting  of  only  a  few  amino  acids 
united  by  the  "peptid  linking."  These  compounds  have  been 
obtained  by  hydrolyzing  protein,  and  also  have  been  built  up 
artificially  in  the  laboratory,  as  already  has  been  indicated  at 
an  earlier  point  in  the  discussion  of  proteins. 

The  final  products  of  hydrolysis,  the  ami7io  acids  are  the  ul- 
timate building  stones  of  which  the  proteins  and  their  simpler 
derivatives  are  built  up.    We  have  seen  that  proteins  are  neces- 


PROTEINS  113 

sary  in  the  food  to  maintain  life,  but  after  all  it  is  really  the 
amino  acids  which  are  needed,  for  in  digestion  the  proteins  are 
split  up  into  amino  acids,  and  these  substances  are  supplied  to 
the  living  cells  of  the  tissues.  Some  of  these  amino  acids  are 
used  to  build  up  new  cell  proteins  or  repair  old  ones,  whereas 
others  are  transformed  into  various  compounds  some  of  which 
undoubtedly  have  most  vitally  important  roles  to  play  in  eon- 
trolling  or  instigating  many  of  the  vital  processes.  Others  of 
the  amino  acids  undoubtedly  are  simply  broken  down  and 
burned  in  the  body  as  fuel,  just  as  are  carbohydrates  and  fats 
generally  speaking.  Some  of  the  important  roles  of  the  amino 
acids  will  be  considered  later  in  connection  with  the  study  of 
metabolism. 


CHAPTER  VI 

SOME  FAMILIAR  FOODSTUFFS— SOME 
IMPORTANT  TISSUES 

Some  Important  Foodstuffs 

The  substances  considered  in  the  preceding  chapters  are  for 
the  most  part  foodstuffs.  It  will  be  worth  while,  however,  to 
consider  some  of  the  familiar  substances  usually  called  ' '  foods. ' ' 
Most  of  these  materials  consist  of  a  mixture,  often  of  several 
of  the  classes  or  individuals  of  the  material  bases.  We  may  ask 
the  question,  ''What  is  a  food?"  In  a  broader  sense  a  food 
may  be  defined  as  any  substance  which  is  required  by  the  body. 
This  would  include  oxj'gen  from  the  air,  water,  and  inorganic 
salts,  in  addition  to  carbohydrates,  fats,  proteins,  etc.  But  some 
substances  which  are  excellent  foods,  are  not  indispensable  to 
the  body,  for  example  sugar.  The  body  tissues  are  constantly 
wearing  out  and  requiring  repair.  Materials  for  such  repair  of 
worn  out  tissues,  or  for  the  building  of  new  ones,  as  in  growth, 
are  obtained  from  foods.  Also,  the  body  is  constantly  doing 
work,  either  external  work,  or  the  work  of  the  heart,  the  diges- 
tive organs,  glands,  etc.  Work  cannot  be  done  without  the 
expenditure  of  energy.  This  energy  is  obtained  by  oxidizing, 
' '  burning, ' '  material  derived  either  directly  or  indirectly  from 
the  foods.  Some  substances  can  be  burned  in  the  body,  but 
still  are  harmful  because  of  a  poisonous  effect  upon  the  cells ; 
such  substances  should  not  be  classed  as  foods.  Summing  up 
the  foregoing  statements,  a  food  may  be  defined  as  a  substance 
which  is  necessary  to  the  body  or  which  furnishes  it  with 
energy  or  building  material,  and  which  is  not  harmful  to  the 
organism. 

Water  and  salts,  which  are  very  important  foods  have  been 

114 


IMPORTANT    FOODSTUFFS,    IMPORTANT    TISSUKS  115 

considered  in  connection  with  the  general  composition  of  the 
body. 

Cooking  and  Preparation  of  Foods. — Some  foods  are  pala- 
table and  digestible  when  raw,  such  as  milk,  fruits,  nuts  and 
some  vegetables.  Other  foods  usually  are  prepared  for  use  by 
cooking.  Proper  cooking  of  food  is  very  important,  both  from 
an  economic  and  from  a  physiological  standpoint.  Inexpensive 
materials,  if  properly  prepared  may  be  quite  as  good  and  de- 
sirable foods  as  more  expensive  materials,  but  even  the  best  of 
materals  may  be  spoiled  and  rendered  unfit  for  use  by  careless 
or  improper  cooking.  In  cooking,  various  changes  are  brought 
about  in  the  foods.  Most  animal  proteins  are  coagulated,  but 
this  does  not  diminish,  and  sometimes  increases  their  digestibil- 
ity. The  closely  packed  fibers  of  "meat  are  loosened  up  so  that 
they  are  more  easily  torn  apart  and  subjected  to  the  action  of 
the  digestive  enzymes.  Vegetable  materials  also  are  softened 
and  made  more  vulnerable  to  the  attack  of  enzymes.  The  taste 
and  appearance  of  food  materials  also  are  improved  in  the  proc- 
ess, and  this  appeal  to  the  appetite  is  of  actual  physiological 
value  in  digestion.  A  further  service  performed  by  proper 
cooking  is  the  destruction  of  living  parasites  of  various  sorts 
Avhich  often  are  found  in  raw  foodstuffs,  especially  if  the  ma- 
terials have  been  exposed  in  provision  shops,  or  have  been  kept 
longer  than  is  wise.  Great  progress  has  been  made  in  recent 
years  in  regulating  the  care  and  quality  of  foodstuffs  and  thus 
in  protecting  the  general  public  from  dangers  arising  from  the 
practices  of  ignorant,  careless,  or  unscrupulous  dealers.  Too 
much  care  and  thoughtfulness  can  scarcely  be  spent  in  the 
proper  choice  and  preparation  of  the  diet. 

Milk. — Milk  is  one  of  the  most  important  natural  foodstuffs. 
It  is  the  natural  and  ideal  food  for  the  growing  young,  and 
comes  very  near  to  being  an  ideal  food  even  for  the  adult.  It 
contains  all  the  necessary  food  substances,  and  in  vciy  nearly 
the  proper  proportions.  If  bread  or  crackers  are  eaten  with 
it,  it  is  an  ideal  food.  The  carbohydrate  of  milk,  lactose,  already 
has  been  discussed.     Cow's  milk  contains  about  4%  lactose,  hu- 


116  PHYSIOLOGICAL    CHEMISTRY 

man  milk  about  6-7%,  Milk  fat,  about  2-4%  in  both  cow's  and 
human  milk  is  characterized  by  its  content  (about  8%  of  the 
total  fats)  of  lower  fatty  acids,  butyric,  caproic,  caprylic  and 
capric.  There  also  are  traces  of  phosphatids.  There  are  at 
least  three  proteins,  (0.7-1.5%  in  human  mlk  and  2.5-4%  in' 
cow's  milk).  Casein  makes  up  about  80%  of  the  total  protein, 
the  remainder  is  mostly  lactalbumin,  with  a  small  amount  of 
lactoglobulin.  Milk  also  contains  a  variety  of  salts  including 
those  of  calcium,  magnesium,  sodium,  potassium  and  iron;  it 
contains  phosphates  and  chlorides.  Milk  contains  about  75% 
water. 

If  milk  is  allowed  to  stand,  bacteria  decompose  lactose  form- 
ing organic  acids.  The  milk  becomes  disagreeable  in  taste,  and  if 
the  process  goes  far  enough  the  casein  is  precipitated,  and  the 
milk  is  said  to  be  sour.  By  properly  sterilizing  or  pasteurizing 
milk,  the  bacteria  are  destroyed,  and  if  it  then  is  kept  in  closed 
vessels  and  not  exposed  to  the  air  it  will  keep  for  a  much  longer 
time  than  otherwise.  Half  emptied  milk  bottles  should  on  no 
account  be  left  uncovered  in  a  room  or  in  the  family  ice  chest, 
as  bacteria  from  the  air,  falling  into  the  milk,  will  thrive  and 
multiply,  and  the  milk  if  used  may  thus  become  a  menace  to 
health. 

When  milk  stands  for  some  time  a  portion  of  the  fat  rises 
to  the  surface.  If  this  layer  is  skimmed  off  the  liquid  is  called 
cream.    Average  cream  contains  18-20%  fat. 

Buttermilk  differs  from  sweet  milk  chiefly  in  its  lower  fat 
content  (about  0.5%  fat),  since  most  of  the  fat  has  been  re- 
moved as  butter.  As  butter  is  usually  made  from  old  cream, 
buttermilk  often  is  sour,  that  is,  contains  some  lactic  acid  pro- 
duced by  the  fermentation  of  lactose. 

Butter. — Butter  is  a  food  of  very  high  fuel  value,  for  it 
contains  80%  or  more  of  fats.  The  composition  of  this  fat 
already  has  been  discussed  (see  under  fats).  Butter  contains 
about  1%  of  protein,  on  an  average  of  3%  salts  and  from  10- 
15%  water.     It  contains  practically  no  carbohydrate.     Oleo- 


IMPORTANT   FOODSTUFFS,    IMPORTANT    TISSUES  117 

margarine,  a  butter  substitute  made  largely  from  beef  fat  also 
has  been  discussed  in  the  chapter  on  fats. 

Cheese. — When  the  caseinogen  of  milk  is  clotted  by  rennin 
it  separates  out  as  a  solid  curd,  which  carries  with  it  much  of 
the  milk  fat.  This  curd  is  cheese.  Cheese  is  a  very  nutritious 
foodstuff.  Different  kinds  of  cheese  vary  greatly  in  composi- 
tion and  food  value.  Average  American  cheese  contains  about 
29%  protein,  36%  fat,  traces  of  carbohydrate,  3.5%  ash  and 
32  %o  Avater. 

Meats. — Meats  are  important  food  substances,  for  they  fur- 
nish large  amounts  of  proteins,  which  are  essential  constitu- 
ents of  the  diet.  Meats  contain  a  variety  of  proteins,  but  the 
chief  protein  of  dead  muscle  is  myosin.  Albumin,  globulins 
other  than  myosin,  albuminoids,  glj^coproteins,  nucleoproteins, 
hemoglobin  derivatives,  and  lecithoproteins  all  are  constitu- 
ents of  meat,  but  in  digestion  all  of  these  substances  are  broken 
up  into  their  constituent  amino  acids,  the  form  in  which  pro- 
tein food  reaches  the  cells  of  the  tissues.  Meat  also  contains 
fats,  and  small  amounts  of  the  carbohydrate  glycogen  or  the 
sugar  derived  from  it.  Most  meats,  with  the  exception  of  bacon 
or  veiy  fat  meats  contain  from  10  to  20%  protein.  Fat  ranges 
from  a  few  per  cent  up  to  60%  in  fat  bacon.  Ash  ranges  from 
about  1%  to  4  or  5%,  water  from  about  7  or  8%  to  68  or  70%. 

Eggs. — Eggs  are  a  very  important  article  of  diet.  In  the 
development  of  the  young  of  birds  they  furnish  the  material 
out  of  which  the  tissues  of  the  chick  are  built  up.  Eggs  con- 
tain (without  the  shell)  about  12%o  protein,  11%  fat,  0.5% 
carbohydrate,  1%  ash,  and  75%  water.  Most  of  the  fat  is  in 
the  yolk.  About  %  of  "t^i®  protein  is  in  the  white  and 
%  in  the  yolk.  About  90%  of  the  total  protein  of  the  white  is 
albumin.  About  61^%  is  globulin.  Vitellin  is  the  chief  yolk 
protein.  All  of  these  substances  have  been  considered  under 
their  respective  groups.  The  yolk  contains  fat  and  also  con- 
siderable quantities  of  lecithin  and  cholesterol. 

Vegetables. — Vegetables  contain  very  high  amounts  of  water 
and  thus  are  low  in  fuel  value.     Some  vegetables  such  as  peas. 


118  PHYSIOLOGICAL    CHEMISTRY 

beans,  and  lentils  contain  only  small  amounts  of  water.  With 
the  exception  of  these  and  a  few  others,  most  vegetables  contain 
but  small  amounts  of  proteins,  very  little  fat  and  varying 
amounts  of  carbohydrates.  As  fuel  substances  therefore  most 
vegetables  are  far  below  meats  and  dairy  products.  Peas,  beans 
and  lentils  are-^  exceptions  to  this  statement.  Vegetables  supply 
the  body  with  some  valuable  salts,  with  some  protein,  fat  and 
carbohydrate,  but  they  are  valuable  also  from  another  stand- 
point. They  contain  cellulose.  Although  cellulose  is  of  little 
food  value,  it  gives  bulk  to  the  food,  and  mechanically  irri- 
tates the  walls  of  the  intestines.  -  This  stimulates  muscular  con- 
tractions which  are  important  for  the  thorough  mixing  of  the 
food  with  the  digestive  juices,  and  its  transportation  along  the 
alimentary  canal.  Fruits  also  for  the  most  part  have  little 
food  value,  but  they  are  wholesome  for  the  same  reason,  and 
they  also  stimulate  the  appetite,  which  is  an  important  factor 
in  proper  digestion.  Nuts  contain  much  protein,  much  fat  and 
often  considerable  carbohydrate  (peanuts  contain  protein  25%, 
fat  38%,  carbohydrate  24%)  and  thus  are  extremely  nutritious. 

Breadstuffs  vary  greatly  in  food  value.  Bread  contains  about 
9%  protein,  very  little  fat,  53%  carbohydrate  and  35%  water. 
Its  fuel  value  is  about  that  of  average  meats,  but  its  protein 
content  is  lower.  Crackers  contain  only  about  5%  water  and 
their  fuel  value  is  about  double  that  of  bread.  Sweet  cakes, 
pastries,  etc.,  usually  have  a  high  fuel  value.  They  often  are 
sufficiently  indigestible,  however,  more  than  to  counterbalance 
their  greater  fuel  content.  Some  of  the  breakfast  foods  are 
valuable  foods  but  many  of  them  are  so  light  that  the  amount 
ordinarily  eaten  has  little  total  fuel  value  aside  from  that  of  the 
sugar  and  cream  which  usually  is  eaten  with  them,  and  which 
may  be  considerable. 

Choice  of  Diet. — Much  has  been  written  upon  the  proper 
choice  of  a  diet.  Undoubtedly  a  mixed  diet  of  wholesome  nour- 
ishing food  in  sufficient  quantity,  but  not  in  extravagant  excess 
is  the  most  desirable  solution  of  the  problem  for  the  average 
healthy   individual.     Different   standards    of   amount   Avill   be 


IMPORTANT   FOODSTUFFS,    IMPORTANT    TISSUES  119 

needed  by  individuals  of  different  size  or  different  habits  of  life. 
For  an  average  sized  (TO  kilos)  city  dweller,  perhaps  9'0  grams 
protein  and  enough  fats  and  carbohydrates  to  make  the  total 
energy  intake  about  2,500  calories  is  about  the  right  amount, 
but  these  figures  will  vary  greatly  with  changing  conditions. 
The  diet  must  contain  certain  unknown  substances  called 
vitamincs,  which  will  be  discussed  in  the  chapter  on  metabolism. 

Some  Important  Tissues 

Although  the  substances  occurring  in  the  different  tissues  have 
been  discussed  under  the  various  groups  of  which  they  are 
members,  it  may  be  useful  from  the  student's  point  of  view  to 
include  here  a  brief  summary  or  survey  of  some  of  the  im- 
portant tissues,  and  to  discuss  some  additional  points  of  inter- 
est. 

Muscle. — The  muscles  make  up  about  %  of  the  body  weight 
in  adults.  They  contain  about  18-20%  protein,  72-78%  water 
and  from  0.15-0.3%  of  glycogen.  If  muscle  tissue  is  subjected 
to  a  very  high  pressure,  a  liquid  known  as  the  plasma  is  squeezed 
out.  This  represents  about  60 %o  of  the  total  muscle  weight. 
The  remaining  material  is  called  the  stroma.  The  plasma  has 
the  power  of  clotting.  The  chief  proteins  of  the  plasma  are 
myosin  and  myosinogen  (or  myogen).  Most  authors  have  con- 
sidered myosinogen  to  be  the  mother  substance  of  myosin.  This 
has  been  disputed,  however ;  it  is  considered  by  some  investiga- 
tors to  be  an  albumin.  The  stroma  contains  a  protein  resembling 
an  albuminoid.  In  addition  to  the  above  substances,  muscle 
contains  various  extractives  and  salts,  such  as  creatine,  urea, 
inosite,  taurine,  and  also  some  lipins.  After  activity,  particu- 
larly if  the  muscle  supply  of  blood  or  oxygen  is  low,  lactic  acid 
is  found.  This  is  believed  to  be  a  product  of  the  partial  oxida- 
tion of  glucose.  The  lactic  acid  may  be  further  oxidized,  or  re- 
built into  something  else. 

The  important  property  of  muscles  is  their  power  of  contract- 
ing. The  process  has  been  extensively  studied,  but  as  yet  it  is 
but  imperfectly  understood.     Heat  is  liberated,  glucose  is  used 


120  PHYSIOLOGICAL   CHEMISTRY 

up  and  CO2  is  given  off.  Possibly  the  swelling  and  shortening 
of  the  muscle  fibers  is  caused  by  the  acid  and  heat.  The  in- 
dividual muscle  fibers  become  shorter  and  thicker.  The  acid 
is  quickly  destroyed,  CO2  is  given  off,  and  relaxation  follows. 
If  the  acid  is  not  removed,  cramps  or  rigor  result.  It  has  been 
suggested  that  the  swelling  of  the  fibers  is  caused  by  taking  up 
water  under  the  influence  of  acid,  since  proteins  swell  in  acid 
solution.     The  question  is  still  unsettled,  however. 

Brain  and  Nerves. — Brain  and  nerve  substance  makes  up  a 
far  smaller  proportion  of  body  weight  than  do  the  muscles,  but 
the  primary  importance  of  this  kind  of  tissue  is  obvious.  The 
brain  and  nerves  are  the  directing  mechanism  which  controls 
the  body  activities.  In  higher  animals,  particularly  in  man 
where  intelligence  is  at  the  maximum,  the  brain  is  relatively 
larger  than  in  lower  forms.  The  composition  of  brain  and  nerve 
tissue  is  thus  of  great  interest.  This  kind  of  tissue  contains 
less  protein  than  the  muscles.  Gray  matter  contains  85-]-% 
water,  white  matter  10-\-%  water.  The  characteristic  fact  in 
the  composition  of  brain  and  nerve  tissue  is  the  presence  of 
large  quantities  of  alcohol-ether  soluble  substances.  These  in- 
clude cholesterol,  phospholipins,  cerebrosides,  but  almost  no  neu- 
tral fat,  possibly  none  at  all.  The  principal  phospholipins  are 
lecithin,  kephalin  and  various  myelins;  of  the  cerebrosides  the 
most  important  are  cerebron,  phrenosin  and  kerasin.  Various 
extractives  also  are  found,  such  as  creatine,  inosite,  purine 
bases,  lactic  acid  and  other  substances.  Caprine  ( cc  NH2 
caproic),  an  amino  acid  occurring  in  brain  protein  has  been 
found  in  no  other  tissue.     Glycogen  is  not  stored  in  the  brain. 

The  brain  and  nerves  show  a  vigorous  oxidative  metabolism, 
producing  COo.  They  are  very  sensitive  to  lack  of  oxygen,  and 
quickly  cease  functioning  properly  if  their  oxygen  supply  is 
cut  off. 

Nothing  is  known  of  the  chemical  changes  involved  in  mental 
activity.  Since  the  lipins  are  present  in  such  large  amount,  it 
is  possible  that  they  are  concerned  in  some  way  in  these  proc- 
esses. 


IMPORTANT   FOODSTUFFS,    IMPORTANT    TISSUES  121 

Bones  and  Teeth. — The  bones  and  teeth,  which  form  the 
solid  structural  portions  of  the  body  are  composed  of  both  or- 
ganic and  inorganic  material.  The  bones  are  living  tissue,  and 
their  cells  wear  out  and  are  repaired  just  as  any  other  cells. 
About  60%  of  bone  tissue  is  organic  material,  largely  the  protein 
collagen.  There  also  is  some  mucoid, — osseomucoid.  In  the  hol- 
low bones  marrow  is  found,  and  this  material  contains  much  fat. 
There  are  two  kinds  of  marrow,  yellow  and  red,  the  red  con- 
taining many  red  corpuscles.  In  the  bones  extensive  amounts 
of  inorganic  material  are  found,  chiefly  calcium  phosphate  and 
carbonate. 

The  teeth  are  composed  of  three  materials, — enamel,  dentine, 
and  cement.  The  enamel  is  the  hardest  substance  in  the  body 
and  contains  only  about  .5%  of  water  and  about  91%  of  calcium 
phosphate.  Dentine  and  cement  are  of  about  the  same  composi- 
tion as  bone. 

Cartilage  contains  collagen,  chondroalbuminoid,  salts  and 
mucoid. 

Connective  Tissue. — There  are  two  chief  types  of  connective 
tissue,  yellow  and  white.  The  white  elastic  tissue  consists 
mainly  of  collagen,  the  yellow  of  elastin.  Both  forms  contain 
mucoid  and  extractives. 

The  Blood. — ^Many  points  in  connection  with  the  blood  have 
been  considered  elsewhere,  chiefly  in  the  discussion  of  hemo- 
globin in  the  chapter  on  proteins.  Some  additional  points 
will  be  considered  here.  The  blood  is  a  fluid  in  which  a  variety 
of  formed  elements,  corpuscles  and  platelets,  are  suspended.  It 
is  of  the  utmost  importance  as  a  circulating  medium  for  it  car- 
ries oxygen,  CO,,  food  materials,  products  of  internal  secretion, 
various  waste  substances,  heat,  salts,  etc.,  to  or  from  the  cells. 
The  blood  is  thus  the  common  carrier  of  the  body,  delivering 
fuel  and  other  supplies  to  the  cells,  and  carrying  away  the 
cell  refuse.  Each  cell  is  bathed  in  an  aqueous  fluid  just  as 
were  the  original  independent  unicellular  organisms  which 
lived  in  the  sea. 

The  general  composition  of  the  blood  is  as  follows  (from 
Mathews)  : 


122  PHYSIOLOGICAL    CHEMISTRY 

Plasma   (the  fluid  portion)  60-70% 

Corpuscles  30  -  40% 

Plasma 

Water  90-92% 
Solids     8-10% 
Proteins  5.5 
Carbohydrates  0.1  -  0.2 

Cholesterol,  fat,  extractives 
Inorganic  subst.  1.0-2.0% 
Red  Corpuscles 

Water  59.2  -  68.7% 
Solids  40.8  -  31.3% 

Hemoglobin  31.7% 
Stroma 

Protein  5.7-6.4% 
Phospholipin  0.37-0.39% 
Cholesterol  0.14-0.17% 
Inorg.   (excl.  Fe)     0.09% 

The  important  constituent  of  the  red  corpuscles  is  hemoglobin. 
This  substance  already  has  been  discussed  in  the  chapter  on 
proteins  (q.v.).  It  carries  oxygen  to  the  cells,  and  assists  in 
carrying  away  COo.  In  the  transportation  of  COo,  other  factors 
are  important,  for  this  substance  is  carried  to  a  considerable 
extent  also  in  the  plasma,  as  sodium  bicarbonate,  carbonate  and 
combined  with  plasma  proteins.  When  COo  passes  from  the 
tissues  into  the  blood  it  makes  the  blood  less  alkaline.  This 
favors  the  liberation  of  O2  from  oxyhemoglobin.  The  reverse 
case  occurs  in  the  lungs. 

The  transference  of  0,  into,  and  of  COg  out  of  the  blood  dur- 
ing its  passage  through  the  lungs  has  been  much  studied  to  de- 
termine whether  it  is  a  process  of  simple  diffusion,  or  depend- 
ent upon  secretory  activities  of  the  cells  of  the  alveolar  epithe- 
lium. It  is  now  believed  that  under  ordinary  conditions  dif- 
fusion is  the  principal,  if  not  the  sole  cause  of  the  passage  of 
gases  to  and  from  the  blood.  It  is  probable,  however  that  in 
times  of  stress  there  may  be  an  active  secretion  of  gases  by  the 
cells  to  supplement  the  ordinary  diffusion. 

The  white  corpuscles  are  true  cells,  and  possess  a  nucleus. 


IMPORTANT   FOODSTUFFS,    IMPORTANT    TISSUES  123 

They  can  move  about,  can  reproduce,  and  have  other  functions 
characteristic  of  living  cells  generally. 

The  red  corpuscles  are  living  but  contain  no  nuclei  in  mam- 
mals. There  are  from  5-6  millions  per  cubic  millimeter  in  nor- 
mal blood,  but  they  may  fall  to  one-half  that  number  in  certain 
diseased  conditions. 

The  platelets  are  structures  derived  probably  from  the  white 
corpuscles,  possibly  also  from  the  red.  They  play  an  important 
role  in  the  clotting  of  the  blood.  It  has  been  reported  that  they 
contain  nuclein,  but  this  has  been  contradicted. 

The  chief  plasma  proteins  are  serum  albumin,  one  or  more 
serum  globulins  and  fibrinogen. 

The  blood  contains  various  enzymes  in  small  amounts,  e.g. 
amylase  (acts  on  glycogen),  a  glycolytic  enzyme  (acts  on  glu- 
cose), lipase,  proteolytic  enzymes  and  an  invertase,  the  amount 
of  which  is  increased  if  cane  sugar  is  injected  or  fed  in  large 
amounts. 

A  reaction  which  has  attracted  much  attention  in  the  last  few 
years  is  knov/n  as  the  Abderhalden  reaction.  This  investigator 
found  that  if  a  foreign  protein  is  injected  into  the  blood, 
enzymes  appear  which  have  the  power  of  breaking  down  the 
albumoses  produced  by  digesting  that  protein  with  cold  HoSO^ 
for  24  hours.  The  serum  of  a  pregnant  woman  will  digest  an 
albumose-peptone  mixture  obtained  from  placental  tissue  in 
this  way.  The  digestion  of  the  material  can  be  followed  by  ob- 
serving the  slight  change  in  optical  activity  of  the  mixture,  or 
by  dialyzing  off  the  amino  acids  produced  and  confirming  their 
presence  by  the  ninhydrin  test.  There  is  some  difference  of 
opinion  as  to  the  value  and  reliability  of  this  test  for  the  diag- 
nosis of  pregnancy. 

Reaction  of  the  Blood. — The  blood  is  almost  neutral — it  is 
alkaline  to  litmus  and  acid  to  phenolphthalein.  Nevertheless, 
much  acid  must  be  added  to  blood  before  any  great  change  in 
its  reaction  is  produced.  This  is  due  to  the  fact  that  it  contains 
NaHCO,,  Na/'O,  and  Na,HPO,,  and  alkali  salts  of  the  blood 
proteins.    Any  strong  acid  will  form  its  sodium  salts,  taking  the 


124  PHYSIOLOGICAL    CHEMISTRY 

sodium  away  from  the  weak  acid  radicles  enumerated  above. 
These  weak  acids  do  not  ionize  to  any  great  extent,  and  the 
reaction  of  the  liquid  remains  near  the  neutral  point.  Acids 
produced  in  pathological  conditions  may  be  neutralized  in  a 
similar  way. 

The  hydrogen-ion  concentration  of  blood  as  determined  by  the 
gas  chain  or  indicator  method  (see  the  discussion  of  HCl  in  the 
chapter  on  gastric  digestion)  is  about  N  X  lO''^-^  to  N  X  10'^-^ 
in  defibrinated  blood  at  18°-20°. 

Osmotic  Pressure. — The  osmotic  pressure  of  the  blood  influ- 
ences the  passage  of  water  to  and  from  the  cells.  It  is  kept 
at  a  uniform  level  by  the  kidneys,  which  excrete  excessive 
amounts  of  salts,  urea,  and  sugar.  These  substances  are  the 
main  factors  in  determining  the  osmotic  pressure  of  the  blood. 
The  average  depression  of  the  freezing  point  in  mammalian 
blood  is  A  =  —0.5°  to  —0.6°  C. 

Coagidation  of  the  Blood. — An  enormous  amount  of  labor 
has  been  expended  to  clear  up  the  mechanism  of  blood  clotting. 
The  results  still  are  far  from  conclusive.  Clotting  is  a  protec- 
tive device  to  prevent  excessive  loss  of  blood  after  injury.  If 
blood  is  drawn  and  allowed  to  stand,  it  sets  to  a  jelly-like  mass. 
On  standing,  a  yellow  liquid  is  pressed  out.  This  is  called  the 
serum.  It  differs  from  plasma  in  that  it  does  not  contain 
fibrinogen,  for  in  clotting,  fibrinogen  is  converted  into  the  insol- 
uble fibrin.  This  fibrin  forms  a  fine  mesh  work  in  which  the 
corpuscles  are  entangled.  Contact  with  foreign  bodies  or  with 
the  injured  tissue  hastens  clotting.  Blood  platelets  and  calcium 
play  a  role  in  the  clotting  process.  In  the  process  of  clotting 
a  new  substance,  thrombin  appears.  A  second  new  substance, 
serum  fibrinogen  also  has  been  reported. 

The  following  summary  of  the  steps  involved  in  clotting  is 
perhaps  the  most  satisfactory  explanation  thus  far  advanced. 
Since  clotting  does  not  occur  normally  in  blood  circulating  in 
the  blood  vessels,  some  one  or  more  of  the  substances  involved  in 
clotting  must  be  absent  from  such  blood,  or  be  held  in  check 
or  in  an  inactive  form.     This  substance   is  considered  to  be 


IMPORTANT    FOODSTUFFS,   IMPORTANT    TISSUES  125 

thrombin,  which  is  present  in  the  blood  as  prothrombin.  Pos- 
sibly prothrombin  is  combined  with  a  substance  "anti- 
thrombin. ' '  When  blood  is  shed,  as  in  injury,  the  blood  platelets 
yield  a  substance  thrombokinase  which  combines  with  anti- 
thrombin,  liberating  the  prothrombin.  The  prothrombin  then 
reacts  with  calcium  to  form  thrombin.  Thrombin  acts  on  fibrino- 
gen, which  is  converted  into  the  insoluble  fibrin. 

Various  things  delay  or  prevent  clotting,  such  as  substances 
which  precipitate  calcium,  strong  salt  solutions,  alkalies,  cool- 
ing, and  a  substance  contained  in  the  head  of  leeches,  also  some 
snake  venoms  and  other  poisons. 

Lymph. — The  lymph  is  a  fluid  much  resembling  blood  plasma 
in  composition.  It  fills  the  spaces  between  the  tissues  and  or- 
gans, and  serves  as  a  medium  through  which  the  interchange  of 
material  between  blood  and  cells  takes  place. 

The  Skin. — The  skin,  hair,  nails,  etc.,  are  made  up  chiefly  of 
the  protein  keratin,  and  contain  also  various  salts.  The  skin 
also  contains  various  pigments,  some  of  those  of  dark  color 
being  classed  as  melanins. 


CHAPTER  VII 
DIGESTION  IN  THE  MOUTH 

General  Purpose  of  Digestion. — The  body  is  by  no  means  a 
permanent  structure  consisting  always  of  the  same  molecules  of 
its  constituents.  It  is  a  living,  changing  mass  of  material,  fairly 
uniform  in  its  percentage  composition,  to  be  sure,  but  constantly 
wearing  out,  and  being  rebuilt  or  repaired.  The  cells  per- 
form much  work, — they  build  up  substances  for  secretion,  they 
produce  heat  by  the  oxidation  of  their  OAvn  constituents  or  mate- 
rials supplied  to  them,  and  they  perform  mechanical  Avork. 
To  do  all  these  things  the  cells  require  fuel,  and  materials  out 
of  which  to  construct  their  products.  Thus  a  li\dng  organism 
must  take  food.  For  purposes  of  producing  heat  or  mechanical 
work  the  materials  required  are  not  restricted  to  particular 
chemical  substances.  It  is  enough  that  the  materials  supplied 
be  such  that  they  can  enter  the  cells,  and  be  "burned"  by 
them.  For  purposes  of  repair  or  building  specific  products  the 
situation  is  different,  however.  Unless  the  body  can  build  up  a 
constituent  such  as  a  given  amino  acid  required  for  the  manu- 
facture of  a  necessary  substance,  that  particular  constituent 
must  be  supplied  in  the  food.  We  are  beginning  to  realize  that 
definite  building  stones  are  demanded  by  the  cells  for  the  con- 
struction of  their  products.  In  some  cases  the  body  itself  is  able 
to  manufacture  the  necessary  building  stones  from  other  ma- 
terials. In  other  cases  it  is  not  able  to  do  so,  and  serious  conse- 
quences result  if  the  required  building  stone  is  not  supplied  in 
sufficient  amounts.  This  is  another  argument  in  favor  of  a 
varied  diet  which  will  be  likely  to  furnish  all  substances  re- 
quired. 

The  materials  of  our  food  correspond  only  in  a  rough  way  to 
the  materials  out  of  which  our  body  is  constructed.     Many  of 

126 


DIGESTION    IN    THE    MOUTH  127 

the  plant  proteins,  for  example,  are  valuable  foods,  although 
they  differ  from  our  )>ody  proteins  in  the  relative  amounts  and 
in  the  arrangement  of  the  different  amino  acids  they  contain. 
If  the  varied  substances  in  our  food  all  could  get  into  the  blood 
stream,  and  into  the  cells,  the  problems  of  the  cells  would  be 
greatly  complicated,  for  they  would  be  obliged  to  deal  with  a 
great  variety  of  proteins,  carbohydrates  and  fats.     Nature  has 
solved  this  problem  by  arranging  for  the  breaking  up  of  the 
various  foodstuffs  into  their  simple  building  stones  in  the  di- 
gestive tract.    It  is  in  the  form  of  these  simple  building  stones,  a 
limited  uniform  list  of  which  is  obtained  in  general  from  all 
the  food  substances,  that  the  food  materials  reach  the  cells.    An 
exception  is  the  case  of  the  fats,  the  building  stones  of  which 
are  rebuilt  for  the  most  part  into  neutral  fat  before  entering  the 
blood.     The  primary  function  of  digestion  thus  is  to  break  up 
the  diverse  constituents  of  the  foods  into  a  fairly  uniform  mix- 
ture of  simple,  diffusible  compounds  which  enter  the  blood  and 
are  presented  to  the  cells  for  their  use.    The  chief  factors  in  this 
breaking  down  process  are  the  enzymes  present  in  the  digestive 
juices.    The  enzymes  and  the  conditions  affecting  their  activities 
already  have  been  discussed.     (See  chapter  on  carbohydrates.) 
Preparation  of  Food. — Chewing. — Proper  cooking  is  of  great 
importance  in  preparing  food,  as  was  pointed  out  in  the  chap- 
ter on  foods.    After  proper  cooking  comes  proper  chewing.    The 
food  should  be  thoroughly  chewed  to  break  it  up  into  small 
fragments  so  that  the  digestive  enzymes  may  have  better  access 
to  its  constituents. 

Saliva. — Secretion,  Amount. — In  the  process  of  chewing,  the 
food  not  only  is  broken  up,  but  it  becomes  mixed  with  the  first 
of  the  digestive  juices,  the  saliva.  The  saliva  is  secreted  by 
three  sets  of  glands,  the  parotid,  submaxillary  and  sublingual, 
and  also  by  small  glands  in  the  mucous  membrane.  The  ma- 
terials secreted  by  the  different  glands  vary,  as  do  also  the 
nature  of  the  stimuli  which  will  cause  a  secretion.  Each  set  of 
glands  is  controlled  by  nerves  from  the  brain  direct,  and  from 
the  sympathetic  system.     A  flow  of  saliva  may  be  produced  by 


128  PHYSIOLOGICAL    CHEMISTRY 

mechanical  irritation  of  the  mouth,  by  reflex  action  upon  stimu- 
lation of  the  end  organs  of  taste  or  smell,  by  purely  psychic  in- 
fluences, such  as  the  thought  or  sight  of  food,  or  by  a  general 
reflex,  as  in  nausea  preceding  vomiting.  Its  flow  may  be  in- 
hibited by  nervous  stress  or  anxiety,  a  fact  well  known  to 
amateur  public  speakers  or  by  those  who  have  experienced  dry- 
ness of  the  throat  in  sudden  fright.  Some  drugs  stimulate, 
others  retard  the  flow  of  saliva.  The  composition  of  the  secre- 
tion is  influenced  by  the  nature  of  the  stimulus  producing  it. 
The  amount  of  saliva  secreted  in  a  day  undoubtedly  varies 
within  wide  limits.  1500  c.c  a  day  has  been  suggested  as  an 
average  amount.  Many  factors  may  greatly  increase  this 
amount  such  as  smoking,  continuous  chewing,  mercury  poison- 
ing, or  various  drugs,  notably  pilocarpine. 

Composition  of  tJie  Mixed  Saliva. — The  mixed  saliva  is  a  thin 
watery  fluid  (99.4%  water)  containing  various  salts,  including 
potassium  sulphocyanate,  and,  as  its  most  important  constitu- 
ents, mucin  and  an  enzyme  ptyalin.  Other  substances  of  vary- 
ing nature  are  present  in  small  amounts.  The  saliva  usually  is 
somewhat  turbid,  due  to  cells  or  other  material,  and  on  stand- 
ing the  turbidity  increases,  due  to  the  precipitation  of  calcium 
carbonate.  It  often  is  thick  and  somewhat  slimy  in  character 
from  the  mucin  present,  most  of  which  is  secreted  by  the  sub- 
maxillary and  sublingual  glands.  Mucin  is  a  glycoprotein  which 
already  has  been  discussed  in  the  chapter  on  proteins.  Saliva  is 
usually  slightly  alkaline  in  reaction,  (H-ion  concentration  about 
2X10"^)  but  so  slightly  that  it  is  acid  to  phenolphthalein.  In 
certain  individuals  the  saliva  is  somewhat  acid.  The  causes  of 
such  a  condition  as  yet  are  but  imperfectly  understood. 

Functions  of  Saliva. — The  saliva  moistens  the  mouth  and  food, 
and  by  reason  of  the  mucin  present,  serves  as  a  lubricating  agent 
to  aid  in  the  manipulation  and  swallowing  of  the  food.  It  also 
cleans  and  preserves  the  teeth,  both  by  washing  away  particles 
of  food  which  otherwise  might  decay,  producing  acids  which 
would  attack  the  teeth,  and  also  by  neutralizing  acids  introduced 
into  the  mouth.    The  saliva  also  performs  an  important  digestive 


DIGESTION    IN    THE    MOUTH  129 

function  by  reason  of  the  enzyme  ptyalin  which  it  contains. 
Ptyalin  is  an  enzyme  which  acts  on  starches.  Little  is  known  of 
its  chemical  nature,  but  evidence  seems  to  indicate  that  it  is 
made  up  of  a  substance  resembling  a  protein  combined  with  a 
carbohydrate  gum.  Perhaps  ptyalin  is  not  a  single  enzyme,  but 
a  mixture  of  two  or  more  enzymes,  each  of  which  is  responsible 
for  one  of  the  steps  in  the  digestion  of  starch.  Ptyalin  is 
secreted  by  the  parotid  glands.  It  acts  best  at  a  temperature  of 
40°-45°  C.  and  is  destroyed  by  heating  rapidly  to  75°  C. 
Ptyalin  acts  best  in  a  very  faintly  acid  solution,  the  optimum 
acidity  being  10-®- ^X  normal.  If  the  acidity  is  sufficient  to  turn 
Congo  red  to  violet  (N  X  10"*)  the  action  of  ptyalin  is  inhibited. 
It  also  will  act  in  a  weakly  alkaline  solution  such  as  the  saliva. 
The  presence  of  some  salt  favors  its  action. 

Starch  is  attacked  by  ptyalin  and  broken  down  into  simpler 
substances.  The  physical  condition  of  the  starch  is  important, 
for  raw  starch  is  digested  only  with  difficulty,  whereas  cooked 
starch  is  digested  very  rapidly  and  completely  under  favorable 
conditions  of  temperature,  acidity,  etc.  Various  products  are 
produced  in  the  breaking  up  of  starch,  first  the  dextrins,  of 
which  there  undoubtedly  are  several,  and  finally  maltose  and 
isomaltose.  The  progress  of  the  decomposition  may  be  followed 
by  testing  small  portions  of  the  liquid  with  iodine.  The  blue 
color  characteristic  of  starch  gives  place  to  a  purplish,  then  a 
red  color,  and  finally  no  color  whatever  is  produced.  The 
products  corresponding  to  these  stages  are  as  follows: 

Color  with  Iodine 

Starch  — >  Amylodextrin  — >  Erythrodextrin  -^  Achroo-dextrin 
blue  blue  red  (no  color) 

-^  Maltose 
(no  color) 

Maltose  undoubtedly  is  split  oK  even  in  the  first  stages  of  the 
process. 

The  saliva  also  contains  small  amounts  of  an  enzyme  maltase, 
which  breaks  up  some  of  the  maltose  into  glucose.     Perhaps,  as 


130  PHYSIOLOGICAL   CHEMISTRY 

already  stated,  there  are  different  enzymes  which  are  responsible 
for  the  different  steps  of  the  decomposition  of  starch. 

Since  the  action  of  ptyalin  is  inhibited  by  concentrations  of 
acid  such  as  are  present  in  gastric  juice,  it  might  be  expected 
that  its  activity  would  last  only  during  the  brief  time  before 
the  food  enters  the  stomach.  It  has  been  shown,  however,  that 
when  a  meal  is  taken,  the  food  forms  a  bolus  or  mass  in  the 
stomach  and  does  not  become  thoroughly  mixed  with  the  acid 
gastric  juice  for  some  time,  perhaps  a  half  hour.  The  ptyalin 
thus  continues  to  act  under  these  conditions  for  some  time  after 
the  food  is  swallowed,  and  the  decomposition  of  starchy  ma- 
terials may  be  considerable.  Ptyalin  is  not  found  in  the  saliva 
of  all  animals.  It  is  missing,  for  example,  in  the  saliva  of  the 
dog,  cat  and  some  others  of  the  earnivora.  Their  natural  food 
is  not  starchy  in  character. 

The  saliva  also  contains  an  enzyme  erepsin  which  has  the 
power  of  splitting  peptids,  but  saliva  does  not  digest  proteins  or 
fats.  Certain  substances  are  excreted  in  the  saliva.  Potassium 
sulphocyanate  is  present  in  small  traces,  but  is  not  known  to 
perform  any  definite  function.  If  a  capsule  containing  potas- 
sium iodide  is  swallowed,  iodide  soon  may  be  demonstrated  in 
the  saliva.  It  is  taken  out  of  the  blood  and  excreted  by  the 
salivary  glands.  Urea,  uric  acid  and  many  other  substances  also 
have  been  found  in  traces  in  the  saliva.  Perhaps  they  merely 
filter  through  from  the  blood. 


CHAPTER  VIII 
DIGESTION  IN  THE  STOMACH 

Importance. — When  the  food  has  been  mixed  with  the  saliva 
and  ground  into  small  pieces  by  the  teeth  it  is  swallowed,  and 
passed  down  the  esophagus  into  the  stomach.  The  stomach  is 
a  muscular,  strong  walled  organ,  which  is  collapsed  or  con- 
tracted upon  itself  when  empty,  but  is  capable  of  being  greatly 
distended.  If  water  is  taken  alone,  it  quickly  runs  through  the 
stomach  into  the  intestine,  but  solid  food  collects  in  a  more  or 
less  solid  mass  near  the  entrance.  It  remains  in  the  stomach 
for  a  considerable  time,  (1-5  hours)  passing  on  little  by  little 
as  it  is  thoroughly  mixed  with  the  gastric  juice.  The  stomach 
thus  serves  primarily  as  a  reservoir  into  which  considerable 
amounts  of  food  may  be  taken  at  a  time.  Since  its  walls  are 
strong  and  muscular  they  are  in  no  danger  of  being  ruptured. 
The  stomach  thus  stands  between  the  more  delicately  constructed 
intestine  and  the  mass  of  food  taken  in  a  normal  meal,  and 
passes  along  the  food  material  in  small  amounts  so  as  not  to 
overtax  the  capacity  of  the  intestine.  However,  the  stomach 
also  fulfils  important  digestive  functions. 

Methods  of  Study. — The  digestive  functions  of  the  stomach 
are  due  to  constituents  of  a  juice  secreted  by  numerous  small 
glands  scattered  in  the  stomach  wall.  The  study  of  this 
juice  and  its  activities  has  received  the  attention  of  scientific 
men  for  a  very  long  time.  One  of  the  chief  difficulties  was  the 
manner  of  obtaining  juice  for  study.  Spallanzani,  working  in 
the  latter  part  of  the  eighteenth  century,  swallowed  food  tied 
up  in  small  linen  bags  which  ultimately  were  passed  with  the 
feces.  By  studying  the  residues  in  the  bags,  the  effects  of  diges- 
tion on  the  food  could  be  observed.  Another  method  consisted  in 
swallowing  a  sponge  tied  to  a  string.     The  sponge  soaked  up 

131 


132  PHYSIOLOGICAL    CHEMISTRY 

gastric  juice,  and  was  then  pulled  out.  Such  methods  were 
obviously  crude,  and  led  to  but  imperfect  results.  At  the  pres- 
ent time,  to  study  gastric  contents  a  light  meal  is  given,  and 
after  a  time  the  stomach  content  is  pumped  out  with  a  stomach 
pump.  This  material  consists  of  gastric  juice  mixed  with  food, 
but  the  method  may  give  data  valuable  for  diagnosis. 

The  first  successful  study  of  gastric  juice  and  the  condi- 
tions causing  its  flow  was  made  possible  by  an  accident  which 
occurred  in  1822  on  the  Island  of  Mackinac.  Alexis  St.  Mar- 
tin, a  Canadian  coureur  du  hois,  was  accidentally  shot  in  the 
abdomen.  The  bullet  passed  through  the  stomach,  and  when 
the  wound  healed,  a  passageway  was  left  from  the  stomach  to 
the  exterior.  St.  Martin  recovered  and  lived  for  a  long  time 
in  perfect  health.  He  came  under  the  care  of  Dr.  William 
Beaumont,  an  American  army  surgeon,  who  recognized  the 
exceptional  opportunity  to  study  the  problems  of  gastric  diges- 
tion, and  made  a  long  series  of  careful  investigations.  Two 
Europeans  developed  the  same  method  of  study  by  producing 
artificial  openings  or  "  fistulas "  into  the  digestive  tract  of  dogs. 
The  Russian,  Pavlov,  has  brought  the  technique  of  the  method 
to  its  highest  stage  of  perfection.  An  incision  was  made  into 
the  stomach,  and  the  cut  sewed  up  in  such  a  way  as  to  produce 
a  small  pouch,  or  "little  stomach"  separated  from  the  main 
stomach  cavity,  but  still  supplied  with  blood  vessels  and  nerves. 
From  this  pouch,  a  silver  tube  led  to  the  exterior.  If  gastric 
juice  was  poured  out  into  the  small  pouch,  it  could  be  drawn  off 
to  the  exterior  and  its  composition  and  properties  studied.  These 
observations  made  on  dogs  have  been  supplemented  by  occa- 
sional investigation  on  human  beings  when  restriction  of  the 
esophagus  or  other  causes  made  it  necessary  to  make  an  open- 
ing into  the  stomach  from  the  exterior.  By  the  use  of  such 
methods  a  large  number  of  investigators  have  studied  the  prob- 
lems of  gastric  secretion  and  digestion.  The  results  of  these 
studies  are  summarized  in  the  following  discussion. 

Causation  of  Flow  of  Gastric  Juice. — The  interior  of  the 
stomach,  when  no  food  is  present,  is  pale  pink  and  velvety  in 


DIGESTION    IN    THE    STOMACH  133 

appearance.  Gastric  juice  does  not  flow  continuously.  There 
are  two  factors  which  cause  a  floAV  to  begin,  nerve  impulses 
and  chemical  stimulation  of  the  glands.  The  former  may  have 
two  origins,  psychic  or  reflex.  The  mere  thought  or  sight  of 
food  may  cause  a  flow  of  gastric  juice.  This  is  known  as  the 
psychic  or  appetite  secretion.  The  presence  of  food  in  the 
mouth  gives  rise  to  a  copious  flow  by  reflex  action.  The  flow 
produced  by  nervous  stimulation  is  large  in  amount,  and  con- 
tinues for  some  time.  It  does  not,  however,  account  for  all  of 
the  gastric  juice  poured  out,  for  if  a  dog  is  allowed  to  swallow 
the  food  which  he  chews,  the  amount  of  juice  obtained  is  greater 
than  when  the  food  is  caused  to  drop  out  of  an  opening  made 
into  the  esophagus.  The  presence  of  the  food  in  the  stomach 
thus  is  responsible  for  a  portion  of  the  secretion.  Some  investi- 
gators, among  them  Beaumont,  have  reported  that  mere  mechan- 
ical stimulation  of  the  stomach  walls  by  means  of  a  feather  or 
a  glass  rod  would  cause  secretion.  Pavlov  beli'eves,  however, 
that  mechanical  stimulation  causes  no  flow.  The  secretion  re- 
sulting from  the  presence  of  food  in  the  stomach  is  not  due  to 
reflex  nervous  impulses  acting  on  the  gastric  glands,  for  it  oc- 
curs even  after  the  nervous  connections  of  the  stomach  have 
been  severed.  All  foods  will  not  cause  a  flow,  therefore  it  can- 
not be  the  result  of  mechanical  stimulation.  It  is  evident  that  a 
chemical  substance  must  be  involved,  which  stimulates  the  gas- 
tric glands  to  action.  Injecting  a  partially  digested  mixture 
into  the  blood  does  not  cause  a  flow.  From  this  and  other 
evidence  is  has  been  concluded  that  by  the  action  of  the  food  on 
the  pyloric  mucous  membrane  a  substance  is  produced  which  is 
given  off  to  the  blood  stream.  When  this  substance,  to  which 
the  name  of  gastrin  has  been  given,  reaches  the  gastric  glands, 
it  incites  them  to  secrete  gastric  juice  which  is  poured  out  into 
the  stomach  cavity.  This  subject  is  further  discussed  in  the 
chapter  on  Intestinal  Digestion.  Some  foods,  such  as  meat 
broth,  produce  this  effect  directly.  Other  foods  do  so  only  after 
they  are  partly  digested.  This  chemical  secretion,  though  smaller 
in  amount  than  the  nervous  secretion,  persists    for    a    much 


134  PHYSIOLOGICAL   CHEMISTRY 

longer  time.  Digestion  in  the  stomach  thus  is  started  by  a 
copious  flow  of  juice  incited  by  nervous  impulses,  either  psychic 
or  reflex.  The  process  is  continued  by  a  flow  which  is  incited 
by  the  presence  of  the  food  itself  in  the  stomach  by  means  of  a 
chemical  substance,  gastrin,  produced  in  the  mucous  mem- . 
brane,  given  off  into  the  blood,  which  upon  reaching  the  gastric 
glands,  incites  them  to  secrete. 

The  amount  of  gastric  juice  secreted  varies  greatly.  It  is 
roughly  proportional  to  the  amount  of  food  eaten.  From  2-3 
liters  a  day  has  been  estimated  as  an  average  amount. 

General  Character  of  the  Secretion. — The  character  and  com- 
position of  the  gastric  juice  vary  with  the  nature  and  amount 
of  food  taken.  Perhaps  this  is  due  to  the  fact  that  all  of  the 
■  glands  producing  gastric  juice  may  not  be  secreting  all  the 
time.  The  juice  first  secreted  has  a  greater  digestive  activity 
than  that  secreted  later,  and  any  sudden  increase  in  the  amount 
of  flow  also  is  "accompanied  by  increased  digestive  power.  The 
digestive  agents  apparently  accumulate  in  the  gland  cells  dur- 
ing rest;  the  first  secretion  would  thus  be  more  active. 

Gastric  juice  from  dogs  or  man  is  a  clear,  watery  fluid,  and 
is  colorless  unless  mixed  with  bile  which  sometimes  gets  into 
the  stomach  through  the  pylorus.  The  specific  gravity  ranges 
from  1.002-1.0059.  It  contains  the  salts  which  are  found  in  the 
blood  serum,  and  some  organic  material.  Its  most  important 
constituents  are  hydrochloric  acid  and  the  enzymes  pepsin, 
rennin,  and  a  lipase.  The  chief  digestive  activity  of  the  stom- 
ach is  on  proteins,  since  pepsin  and  rennin  both  act  on  that 
group. 

Hydrochloric  Acid. — The  first  stage  of  the  digestion  of  pro- 
teins in  the  stomach  consists  in  their  conversion  into  acid  meta- 
protein  by  the  action  of  the  hydrochloric  acid  present  in  the 
gastric  juice.  Variations  in  the  amount  of  acid  occur,  and  if  its 
concentration  departs  sufficiently  from  the  normal,  grave  diges- 
tive disturbances  result.  Variations  in  the  amount  of  hydro- 
chloric acid  accompany  certain  pathological  conditions.  For 
these  reasons,  the  acid  of  the  gastric  juice  long  has  been  the  sub- 


DIGESTION    IN    THE    STOMACH  135 

ject  of  much  study.    The  total  acidity  of  the  gastric  contents  is 
due  to  the  sum  of  several  factors, — free  hydrochloric  acid,  hydro- 
chloric acid  combined  with  protein,  acid  salts  and  small  amounts 
of  organic  acids.     It  may  be  determined  as  follows.     The  pa- 
tient, in  the  morning,  when  the  stomach  is  empty,  is  required 
to  eat  a  "test  meal,"  which  consists  of  a  cup  of  tea  and  a  piece 
of  toast,  or  some  other  simple  articles  of  diet.     At  the  end  of  a 
specified  time,  for  example  forty-five  minutes  or  one  hour,  the 
patient  is  made  to  vomit,  or  his  stomach  is  pumped  out  with  a 
stomach  pump.     A  measured  amount  of  the  unfiltered  stomach 
contents  is  titrated  with  N/10  NaOH  using  phenolphthalein  as 
indicator.     This  indicator  is  sensitive  to  such  small  amounts  of 
hydrogen-ions  (thus  to  such  weak  acidity)  that  the  method  will 
yield  results  corresponding  to  the  total  acidity  from  all  factors. 
100  c.c.  of  normal  gastric  contents  should  require  about  74  c.c. 
of  N/10  NaOH  to  neutralize  it.    By  the  use  of  other  indicators 
which  are  less  sensitive  to  weak  acids,  it  is  possible  to  decide 
whether  all  of  the  acidity  is  due  to  free  hydrochloric  acid,  or 
part  of  it  to  hydrochloric  acid  combined  with  protein  or  to 
weaker  acids.     Grunzberg's  and  Toepfer's  reagents  are  the  best 
indicators  to  determine  free  hydrochloric  acid.     It  is  obvious 
that  a  large  amount  of  a  weak  acid  will  produce  as  high  a  con- 
centration of  hydrogen-ions  as  a  small  amount  of  a  strong  acid. 
For  this  reason  the  above  methods,  and  other  similar  methods 
for  determining  the  various  factors  making  up   total   acidity 
are   inaccurate.     The   accurate  determination   of  hydrogen-ion 
concentration  is  made  by  the  gas  chain  method,  a  process  which 
is  too  difficult  for  the  average  physician.     The  following  method 
gives  approximately  correct  results.    It  consists  in  using  Gunz- 
berg's  reagent,  which  contains  2  g.  phlorglucin,  and  1  g.  vanil- 
lin to  100  c.c.  alcohol.     The  reagent  should  be  kept  in  the  dark. 
A  drop  of  the  reagent  on  a  white  plate  is  dried  on  the  water 
bath.    A  drop  of  the  juice  to  be  tested  is  added  to  the  yellow 
spot.     On  warming,  if  free  hydrochloric  acid  is  present  a  red 
color  develops.     By  diluting,  a  point  will  be  found  where  the 
liquid  just  gives  the  reaction.     At  this  point  the  concentration 


136  PHYSIOLOGICAL    CHEMISTRY 

of  HCl  is  0.0004N.  AVeaker  acid  gives  no  color.  The  results 
of  this  test  correspond  fairly  well  with  those  obtained  by  more 
accurate  methods.  In  pure  gastric  juice  most  of  the  hydro- 
chloric acid  is  free.  Total  acidity  as  determined  with  phenol- 
phthalein,  and  free  hydrochloric  acid  as  detemiined  with  Gunz- 
berg's  reagent  agree  closely.  If  food  is  present  however,  the 
free  hydrochloric  acid  is  greatly  reduced  since  much  of  the  acid 
is  combined  with  protein. 

Human  gastric  juice  contains  on  an  average  0.2%  HCl. 
(That  of  dogs  contains  about  0.6%  HCl).  The  concentration 
of  H-ions  runs  from  10"^  to  7X10"^  N. 

In  diseases  the  amount  of  hydrochloric  acid  may  vary  con- 
siderably. A  condition  in  which  the  amount  is  abnormally  high 
is  spoken  of  as  hyperacidity  (or  hyperchlorhydria).  This  is 
usually  observed  in  eases  of  gastric  ulcer  and  in  neurosis.  If 
the  acidity  is  below  normal  it  is  spoken  of  as  hypoacidity  (or 
hypochlorhydria).  This  condition  often  is  observed  in  cases  of 
cancer  of  the  stomach  or  other  regions.  Sometimes  no  acid  at 
all  is  secreted.  This  condition  is  called  anacidity  (or  achlor- 
hydria).    A  normal  condition  is  called  euchlorhydria. 

Source  of  the  Hydrochloric  Acid. — The  hydrochloric  acid 
is  secreted  mainly  by  glands  in  the  fundus  end  of  the  stomach. 
It  is  not  stored  in  the  cells  previous  to  secretion,  and  probably 
is  not  even  secreted  directly  by  the  cells,  but  is  formed  from  a 
secreted  substance.  The  actual  mechanism  is  a  matter. of  doubt. 
Perhaps  it  is  secreted  as  an  ester,  or  is  formed  from  ammonium 
chloride  or  some  other  chlorine  compound.  The  source  of  the 
chlorine  is  evidently  the  sodium  chloride  of  the  blood. 

The  functions  of  the  hydrochloric  acid  are  various.  First, 
as  we  already  have  seen,  it  dissolves  the  proteins  of  the  food, 
converting  them  into  acid  metaprotein.  It  plays  an  important 
part  in  digestion  of  proteins  by  pepsin,  however,  for  this  en- 
zyme is  secreted  in  an  inactive  form,  and  becomes  active  only 
under  the  influence  of  the  hydrochloric  acid.  An  unimportant 
phase  of  the  function  of  the  hydrochloric  acid  is  in  connection 
with  its  action  on  saccharose.    This  disaccharide  is  easily  hydro- 


DIGESTION    IN    THE    STOMACH  137 

lyzed  by  dilute  hydrochloric  acid,  and  it  is  probable  that  this 
process  takes  place  at  least  to  some  extent  in  the  stomach.  The 
hydrochloric  acid  also  kills  bacteria  and  parasites  in  the  food, 
and  thus  protects  the  body  from  their  attack. 

Enzymes  of  the  Gastric  Juice. — Three  important  enzymes 
are  secreted  in  the  gastric  juice,  pepsin,  rennin  and  a  lipase. 

Pepsin  is  formed  in  glands  of  the  stomach  wall,  particularly 
in  the  fundic  region  but  it  exists  there  probably  in  an  inactive 
form  called  pepsinogen,  which  becomes  active  only  after  secre- 
tion. Pepsinogen  is  stored  up  during  the  period  of  inactivity 
between  times  of  secretion.  The  transformation  of  pepsinogen 
into  pepsin  is  brought  about  by  the  hydrochloric  acid  of  the 
gastric  juice.  Little  is  known  of  the  nature  of  pepsin.  Prob- 
ably it  is  similar  in  nature  to  the  proteins,  or  perhaps  only 
attached  to  a  protein,  but  this  is  still  uncertain. 

Pepsin,  which  digests  proteins,  acts  best  in  weak  acid  solu- 
tion. For  pepsin  from  man,  the  most  favorable  acidity  is  about 
0.3%  hydrochloric  acid  or  a  hydrogen-ion  concentration  of 
about  1.7X10-'N.  to  oXlO'^N.  At  lO'^N.  digestion  almost  stops. 
The  most  favorable  acidity  for  pepsin  corresponds  closely  with 
the  normal  acidity  of  the  gastric  juice  of  the  animal  from  which 
the  pepsin  is  obtained.  Other  acids  may  be  used  in  place  of 
hydrochloric,  but  they  are  not  so  favorable.  Pepsin  is  very 
sensitive  even  to  slight  amounts  of  alkali,  and  is  rendered  in- 
active if  its  solution  is  made  alkaline.  For  the  action  of  pepsin 
it  is  not  necessary  that  the  hydrochloric  acid  be  free.  If  the  acid 
is  combined  with  the  protein  acted  upon,  a  neutral  pepsin  solu- 
tion will  digest  the  protein  quite  readily.  Hydrochloric  acid 
alone  will  digest  proteins,  but  much  more  slowly  than  pepsin 
and  acid  together. 

There  are  various  methods  for  estimating  the  activity  of  a 
pepsin  solution.  The  use  of  iMett's  tubes  is  one  of  the  most 
satisfactory.  Egg  white  is  drawn  up  into  narrow  glass  tubes 
and  coagulated  by  heating.  The  tubes  then  are  cut  into  short 
lengths  (ca.  2  cm.)  and  suspended  in  the  enzyme  solutions.  These 
are  placed  in  the  incubator  for  24  hours.    The  amount  of  diges- 


138  PHYSIOLOGICAL    CHEMISTRY 

tion  of  egg  white  bears  a  relationship  to  the  digestive  power  of 
the  solution.  The  length  of  the  column  of  egg  white  digested 
is  proportional  to  the  square  root  of  the  amount  of  the  enzyme. 
The  products  produced  by  the  digestion  of  the  protein  inhibit 
the  digestive 'action  of  the  pepsin,  so  the  method  is  not  an  ideal 
one,  but  it  is  useful  for  obtaining  comparative  data. 

Products  of  Peptic  Digestion.- — Pepsin-hydrochloric  acid  at- 
tacks the  proteins  and  breaks  them  up  into  fragments.  The  acid 
metaprotein  first  breaks  into  proteoses  of  varying  degrees  of 
complexity.  These  are  again  broken  into  products  which  are  not 
precipitated  by  saturation  with  ammonium  sulphate, — the  pep- 
tones. It  is  probable  in  fact  that  peptones  also  are  produced 
directly  from  the  protein  molecule  in  the  first  stages  of  hydro- 
lysis. There  has  been  much  discussion  as  to  whether  or  not 
amino  acids  are  split  off  by  pepsin.  "Weight  of  evidence  seems 
to  indicate  that  pepsin  does  not  split  off  amino  acids,  and  that 
the  presence  of  these  substances  in  mixtures  digested  by  an  ex- 
tract of  the  stomach  wall  was  due  to  another  enzyme,  called 
erepsin,  or  to  still  other  agencies.  The  proteoses  and  peptones 
produced  by  the  action  of  pepsin  vary  greatly  in  their  composi- 
tion and  properties,  as  might  well  be  expected  on  considering  the 
extremely  complex  nature  of  the  protein  from  which  they  are 
produced. 

The  ultimate  fate  of  pepsin  is  a  matter  of  interest,  of  which 
little  is  known.  It  probably  is  largely  destroyed  in  the  intestine, 
but  a  portion  undoubtedly  is  absorbed  into  the  blood.  What 
happens  to  this  portion  is  not  known,  but  a  part  at  least  is  ex- 
creted in  the  urine.  Perhaps  the  remainder  is  destroyed  by 
agents  in  the  blood. 

Rennin. — Gastric  juice,  or  the  water  extract  of  the  stomach 
mucosa  of  a  young  animal  contains  an  enzyme  rennin,  which 
is  instrumental  in  clotting  milk.  For  this  clotting,  calcium  also 
is  necessary.  The  process  is  belicA^ed  to  consist  in  the  transforma- 
tion of  the  soluble  protein  caseinogen  into  a  derivative  called 
paracasein.  In  this  process  an  "albumose"  or  ''whey  protein" 
is  split  off.    The  calcium  salt  of  caseinogen  is  soluble,  but  that  of 


DIGESTION   IN    THE   STOMACH  139 

paracasein  is  insoluble.  When  rennin  has  transformed  the  ease- 
inogen  into  paracasein,  the  calcium  salt  of  the  latter  precipitates, 
and  the  milk  is  said  to  be  clotted.  Removal  of  the  calcium  by 
precipitation  as  oxalate  will  prevent  clotting,  but  will  not  inter- 
fere with  the  formation  of  paracasein.  When  this  has  been 
formed  b}''  the  action  of  rennin  in  oxalated  milk,  the  rennin  may 
be  destroyed  by  boiling.  Now,  on  adding  a  soluble  calcium  salt 
to  the  milk  it  will  coagulate,  showing  that  the  transformation  of 
cascinogen  into  paracasein  has  taken  place  in  the  absence  of 
calcium. 

Are  pepsin  and  rennin  identical?  Not  only  does  gastric  juice 
clot  milk,  but  extracts  of  a  very  large  number  of  substances  con- 
taining proteolytic  enzymes  show  a  similar  behavior.  The  ques- 
tion has  thus  arisen,  are  pepsin  and  rennin  identical,  or  are  they 
different  enzymes  ?  This  question  is  still  in  dispute.  The  trend 
of  evidence  seems  to  indicate,  however,  that  they  are  not  identi- 
cal. Solutions  have  been  prepared  which  showed  peptic  but  not 
rennin  action,  and  also  the  reverse  is  true.  Also,  peptic  digestion 
takes  place  only  in  acid  solution,  whereas  rennin  will  act  also 
in  neutral  or  weakly  alkaline  solution.  Pepsin  itself  undoubt- 
edly can  clot  milk,  however.  There  is  still  no  uniformity  of 
opinion  as  to  whether  the  two  enzymes  are,  or  are  not  identical, 
although  as  already  stated,  opinion  is  tending  toward  the  latter 
view. 

The  value  of  rennin  to  the  young  animal  depends  on  the  fact 
that  by  clotting,  the  chief  protein  of  the  milk  is  retained  in  the 
stomach  instead  of  running  on  through  into  the  small  intestine. 
It  is  thus  subjected  to  the  action  of  the  enzyme  pepsin. 

Gastric  juice  contains  a  lipase,  which  acts  on  fats.  The  action 
is  not  extensive  however,  except  in  the  case  of  emulsified  fats, 
such  as  those  of  milk,  which  are  digested  to  a  considerable  ex- 
tent. 

The  contents  of  the  small  intestine  may  occasionally  regurgi- 
tate into  the  stomach.  Ordinarily  this  takes  place  only  to  a 
slight  extent.    In  this  way  the  digestive  enzymes  of  the  intestine 


140  •  PHYSIOLOGICAL    CHEMISTRY 

may  get  into  the  stomach  and  exert  their  characteristic  activities 
there. 

The  stomach  wall  is  not  digested  by  the  gastric  juice  because 
the  hydrochloric  acid  cannot  pass  into  the  cells  of  the  mucous 
membrane.  These  cells  also  undoubtedly  contain  antienzymes 
which  counteract  the  effect  of  pepsin,  and  thus  protect  the  tis- 
sues. After  death  however,  or  if  the  blood  supply  to  a  given 
area  is  shut  off,  the  walls  of  the  stomach  are  attacked  and 
digested. 

Passage  of  the  Food  Into  the  Intestine. — The  food  is  carried 
along  the  stomach  by  waves  of  muscular  contraction.  The  en- 
trance from  the  stomach  into  the  small  intestine  is  guarded  by  a 
ring  of  contractile  tissue  which  is  closed  ordinarily.  When  acid 
comes  in  contact  with  the  stomach  side  of  this  ring,  as  it  does 
when  the  food  has  been  thoroughly  mixed  with  gastric  juice,  the 
sphincter  relaxes  and  allows  a  small  portion  of  the  stomach  con- 
tents or  ''chyme"  to  pass  into  the  small  intestine.  When  this 
acid  mixture  comes  in  contact  with  the  wall  of  the  small  intes- 
tine, the  sphincter  closes  again.  Thus  the  chyme  is  passed  into 
the  small  intestine  in  small  portions. 

Gastric  digestion  does  not  complete  the  disintegration  of  the 
foodstuffs.  It  only  starts  the  process,  and  prepares  the  food  for 
further  digestion  by  the  enzymes  in  the  intestine.  In  fact  the 
stomach  can  be  removed  without  causing  death.  In  such  case, 
only  small  amounts  of  food  can  be  taken  at  a  time,  and  its 
character  must  be  carefully  regulated. 


CHAPTER  IX 
DIGESTION  IN  THE  INTESTINE 

General. — Digestion  in  the  mouth  and  stomach,  although  val- 
uable in  beginning  the  breaking  down  of  the  foodstuffs,  does  not 
fit  the  greater  part  of  the  food  for  utilization  by  the  body.  The 
final,  and  by  far  the  most  extensive  portion  of  the  task  falls  to 
the  intestine. 

When  the  acid  chyme  passes  into  the  duodenum,  or  upper  part 
of  the  small  intestine  it  becomes  mixed  with  alkaline  digestive 
juices.  Since  pepsin  is  very  sensitive  to  alkali,  when  this  occurs 
the  peptic  digestion  stops.  "We  now  know,  however,  that  the 
contents  of  the  small  intestine  may  remain  acid  or  neutral  for 
some  time.  The  digestive  activity  of  pepsin  may  thus  continue 
for  a  time  after  the  chyme  has  left  the  stomach. 

There  are  three  important  digestive  juices  secreted  into  the 
intestine,  the  pancreatic  juice,  the  bile,  and  the  succus  entericus 
or  intestinal  juice.  The  methods  employed  in  studying  digestion 
in  the  intestine  are  in  the  main  similar  to  those  used  for  gastric 
digestion, — such  as  the  preparation  of  fistulas  or  openings  into 
the  intestine,  removal  of  duodenal  contents  by  means  of  a  tube 
passed  down  the  esophagus  and  through  the  stomach,  and 
various  other  devices. 

Pancreatic  Juice 

General.— The  pancreatic  juice,  or  "external  secretion"  of 
the  pancreas,  is  produced  by  this  gland,  Avhich  lies  along  the 
duodenum  or  close  to  it,  and  empties  its  secretion  by  two 
main  ducts  and  sometimes  also  smaller  ones.  The  opening 
of  the  pancreatic  duct  in  man  lies  9-10  cm.  below  the 
pylorus.  The  amount  of  juice  secreted  varies  with  the 
nature  of  the  food.     It  has  been  estimated  to  average  500-800 

141 


142  PHYSIOLOGICAL   CHEMISTRY 

c.c.  per  day.  The  secretion  is  continuous  in  herbivora,  whose 
intestines  normally  are  well  filled.  In  man,  however,  the  secre- 
tion is  intermittent,  and  it  is  poured  out  in  large  amount  when 
the  acid  chyme  is  passed  into  the  duodenum  from  the  stomach. 

Mechanism  of  Flow. — The  factors  causing  a  flow  of  pancreatic 
juice  have  been  much  studied.  It  became  evident  that  the  action 
of  the  acid  chyme  on  the  walls  of  the  duodenum  causes  a  copious 
flow.  After  severing  the  nervous  connections  of  the  pancreas, 
the  introduction  of  acid  into  the  duodenum  still  causes  a  flow 
of  juice.  Apparently  it  is  not  caused  by  reflex  nervous  stimula- 
tion alone.  It  was  thought  that  perhaps  the  stimulation  is  due 
to  a  local  reflex,  but  Bayliss  and  Starling  showed  that  an  acid 
extract  of  the  walls  of  the  duodenum,  if  neutralized  and  injected 
into  the  blood  stream  causes  the  pancreas  to  secrete.  Apparently 
then,  the  action  of  the  acid  chyme  on  the  walls  of  the  duodenum 
causes  a  substance  to  be  given  off  into  the  blood  stream.  This 
substance,  when  it  reaches  the  pancreas,  causes  it  to  secrete. 
The  substance  has  been  given  the  name  "secretin."  Substances 
of  this  nature  undoubtedly  are  formed  by  many  tissues  or 
organs  in  the  body  and  sent  off  by  way  of  the  blood  as  chemical 
messengers  to  arouse  activity  in  some  other  organ  or  tissue.  To 
these  substances,  as  yet  of  unknown  constitution,  the  name 
hormone  (derived  from  a  Greek  word  meaning  "I  arouse  to  activ- 
ity") has  been  given.  An  example  of  such  a  substance  already 
has  been  cited  in  connection  with  gastric  secretion.  The  final 
proof  of  the  existence  of  intestinal  secretin  was  obtained  by 
making  a  cross  circulation  between  two  dogs  so  that  blood  from 
one  also  circulated  in  the  other.  Acid  was  placed  in  the  duodenum 
of  one  dog,  and  the  pancreases  of  both  began  to  secrete,  showing 
that  the  stimulating  substance  had  been  carried  in  the  blood. 

It  is  very  probable  that  secretin  is  not  responsible  for  the 
entire  secretion  of  the  pancreas,  but  that  a  reflex  stimulation 
from  the  walls  of  the  duodenum  also  comes  into  play. 

Composition  of  Pancreatic  Juice. — Human  pancreatic  juice  is 
a  water-clear  liquid,  alkaline  in  reaction  since  it  contains  sodium 
carbonate.    It  requires  from  10-15  c.c.  N/10  HCl  to  neutralize 


DIGESTION    IN    THE   INTESTINE  143 

100  e.c.  of  juice,  using  litmus  as  indicator.  The  concentration 
of  hydroxyl-ions  is  about  .0001  N.  It  contains  protein  in  quan- 
tities sufficient  to  cause  a  turbidity  on  boiling,  and  several 
enzymes,  chiefly  trypsin,  lipase,  amylase,  nuclease,  maltase  and, 
at  least  in  young  animals,  a  lactase. 

Trypsin. — Trypsin  is  a  proteolytic  enzyme.  When  secreted 
by  the  gland  it  has  very  little  digestive  action,  but  if  pancreatic 
juice  is  mixed  with  the  secretion  of  the  walls  of  the  small  intes- 
tine, it  becomes  very  much  more  active,  and  digests  proteins 
vigorously.  This  fact  is  generally  believed  to  indicate  that  tryp- 
sin is  secreted  in  an  inactive  form,  called  trypsinogen,  which  is 
converted  into  active  trypsin  by  a  substance,  "  enterokinase, " 
in  the  intestinal  secretion.  The  nature  of  the  activation  is  not 
understood.  Perhaps  trypsin  is  liberated  from  some  other  sub- 
stance with  which  it  is  combined.  Some  authors  report  also 
that  the  bile  has  a  favorable  action  on  tryptic  digestion.  It  has 
been  reported  that  some  other  substances  have  the  power  of 
activating  trypsinogen. 

There  is  some  difference  of  opinion  as  to  the  most  favorable 
reaction  for  tryptic  digestion,  which  is  usually  stated  to  be  a 
slightly  alkaline  reaction.  It  is  certain,  however,  that  trypsin 
will  act  in  alkaline,  neutral  or  even  faintly  acid  solution.  The 
reaction  of  the  duodenal  contents  varies  in  fact,  being  at  first 
acid,  until  it  is  neutralized  or  made  alkaline  by  the  various 
digestive  juices  poured  into  the  intestine. 

Activated  trypsin  attacks  most  proteins  very  vigorously, 
breaking  them  down  into  proteoses,  peptones,  peptids  and  even 
amino  acids.  The  digestion  is  by  no  means  complete,  however, 
and  there  still  are  many  of  the  partially  digested  products.  The 
digestion  is  more  vigorous  and  far  reaching  than  that  of  pepsin, 
and  differs  from  it  also  in  the  fact  that  it  will  take  place  in 
alkaline  solution.  The  previous  action  of  pepsin  on  certain  pro- 
teins appears  to  make  them  more  vulnerable  to  the  attack  of 
trypsin. 

The  activity  of  a  trypsin  solution  may  be  studied  by  methods 


144  PHYSIOLOGICAL    CHEMISTRY 

similar  to  those  used  for  studying  peptic  activity,  e.g.,  Mett's 
method,  etc. 

Pancreatic  juice  also  contains  an  enzyme  called  erepsin,  which 
acts  mainly  on  the  simpler  digestion  products,  proteoses,  pep- 
tones and  peptids,  breaking  them  down  into  amino  acids.  Erep- 
sin  acts  also  on  some  proteins,  such  as  casein.  Thus  the  final  stage 
in  the  intestinal  digestion  of  proteins  is  brought  about  by  the 
erepsin  of  the  intestinal  juice,  or  "succus  entericus."  An  erep- 
sin  also  is  present  in  the  intestinal  mucosa  and  in  fact  in  most  tis- 
sues. Erepsin  reduces  most  of  the  protein  digestive  products  to 
the  amino  acid  stage,  the  form  in  which  they  enter  the  blood. 

It  is  interesting  that  the  kind  of  amino  acids  present  and  their 
arrangement  are  important  factors  in  determining  whether  or 
not  a  given  polypeptid  will  be  digested  by  erepsin. 

Rennin. — Pancreatic  juice  has  the  power  of  clotting  milk,  a 
"rennin"  action.  This  is  thought  by  some  authors  to  be  due  to 
the  pancreatic  erepsin. 

Action  on  Fats. — Steapsin. — Pancreatic  juice  has  the  power 
of  emulsifying  and  splitting  fats.  If  mixed  with  neutral  olive 
oil,  the  mixture  quickly  becomes  acid.  An  enzyme  called 
''steapsin,"  a  lipase,  splits  the  neutral  fat  into  glycerine  and 
fatty  acids.  Steapsin  is  believed  to  be  produced  by  the  cells  of 
the  pancreas.  It  is  by  far  the  most  important  of  the  fat  digest- 
ing enzymes.  It  acts  best  in  a  weakly  alkaline  solution,  the 
optimum  hydrogen-ion  concentration  being  JST  X  "^-  In 
stronger  alkali,  or  in  weak  acid  solution  its  activity  is  greatly 
reduced.  Bile  has  a  very  favorable  effect  upon  the  activity  of 
steapsin.  This  is  partly  due  to  the  fact  that  bile  emulsifies  the 
fats  and  thus  makes  them  more  accessible  to  the  action  of 
steapsin,  but  this  does  not  entirely  explain  the  favorable  effect 
of  bile.  The  bile  salts  evidently  are  the  bile  constituents  con- 
cerned. The  liberated  fatty  acids  combine  in  part  with  the 
alkali  present  to  form  soaps. 

Action  on  Starches. — Amylase  or  "  Amylopsin. " — The  pan- 
creatic juice  contains  an  enzyme  ''amylopsin"  which  acts  on 
starch  and  glycogen,  splitting  them  into  the  dextrins  and  finally 


DIGESTION    IN    THE   INTESTINE  145 

maltose  and  isomaltose  in  much  the  same  way  as  the  ptyalin  of 
saliva  splits  starch.  Starches  which  escape  salivary  digestion,  or 
starch  and  glycogen  eaten  by  carnivora  whose  saliva  contains  no 
ptyalin  thus  are  digested  in  the  intestine.  It  has  been  suggested 
that  perhaps  amylopsin  is  really  a  mixture  of  two  or  more 
enzymes,  w^hich  are  responsible  for  different  phases  of  the  split- 
ting of  starch  into  the  disaccharide  maltoses,  one  enzyme  taking 
the  material  as  far  as  the  dextrin  stage,  the  other  converting  dex- 
trins  into  maltose  and  isomaltose.  The  bile  seems  to  have  little 
or  no  effect  upon  the  activities  of  amylopsin.  The  final  stage  of 
the  digestion  of  starch  is  due  to  an  enzyme  maltase  which  splits 
maltose  into  glucose.  Pancreatic  juice  of  young  animals  also 
contains  a  lactase  which  splits  lactose.  This  disappears  as  the 
animal  grows  older  unless  milk  still  forms  a  part  of  the  diet  as 
in  man,  pigs,  and  some  other  animals.  Pancreatic  juice  has 
been  reported  to  contain  a  nuclease  which  acts  on  nucleic  acid, 
and  splits  it  into  its  component  parts. 

The  Bile 

Causes  of  Flow.  Amount. — The  bile,  secreted  by  the 
liver  into  the  gall  bladder  is  poured  out  into  the  duodenum 
by  way  of  the  bile  duct.  It  is  produced  continuously  by  the 
liver,  but  its  flow  from  the  gall  bladder  into  the  duodenum  is 
intermittent.  The  mechanism  by  which  the  secretion  of  bile  into 
the  digestive  tract  is  controlled  is  little  understood.  The  action 
of  the  acid  chyme  upon  the  walls  of  the  duodenum  seems  to  be 
one  of  the  factors  concerned.  Probably  this  is  due  to  hormone 
action. 

The  amount  of  bile  secreted  by  the  liver  of  man  in  the  course 
of  a  day  has  not  been  determined  accurately  under  normal  con- 
ditions. 550  c.c.  per  day  has  been  suggested  as  a  possible  amount. 
It  may  be  less  than  this,  but  in  all  probability  the  amount  is 
greater.  Observations  on  this  point  have  been  made  in  cases  of 
fistulae,  but  since  the  bile  under  these  conditions  does  not  enter 
the  intestine,  its  neutralizing  effect  on  the  acid  chyme  is  lost; 
this  would  tend  to  increase  the  formation  of  secretin.    But  bile 


146  PHYSIOLOGICAL    CHEMISTRY 

itself,  when  it  enters  the  intestine  is  said  to  increase  the  bile 
secretion.     The  point  is  thus  still  uncertain. 

Composition.  Function. — Human  bile  is  a  clear,  watery,  yel- 
low, brown  or  greenish  liquid  as  it  is  poured  into  the  gall  blad- 
der. Here,  however,  it  becomes  somewhat  more  viscous,  due  in 
part  at  least  to  the  addition  to  it  of  mucinous  material  from  the 
mucous  membrane  of  the  gall  bladder  and  biliary  passages.  Bile 
has  a  bitter  taste,  is  usually  somewhat  alkaline  in  reaction  and 
contains  a  variety  of  substances,  among  them  bile  pigments,  bile 
salts,  cholesterol,  a  mucinous  material,  inorganic  substances  and 
many  other  things.  Although  the  bile  itself  does  not  contain 
digestive  enzymes,  at  least  in  any  important  amount,  it  is  of  the 
greatest  importance  in  the  processes  of  intestinal  digestion.  This 
is  especially  true  in  the  case  of  fat  digestion.  The  addition  of 
bile  to  a  mixture  of  olive  oil  and  pancreatic  juice  increases  the 
amount  of  oil  digested  several  fold.  (5-10  times).  There  has 
been  much  discussion  as  to  the  constituent  of  bile  responsible 
for  this  effect.  Latest  opinions  ascribe  it  to  the  action  of  the 
bile  salts,  sodium  glycocholate  and  taurocholate.  There  also 
are  conflicting  reports  as  to  the  effect  of  bile  on  the  action  of 
trypsin  and  amylase,  but  probably  the  former  is  not  affected,  and 
the  latter  slightly  if  at  all.  The  function  of  the  bile  in  connec- 
tion with  absorption  will  be  discussed  later. 

Bile  tends  to  reduce  putrefaction  in  the  intestine.  It  is  not 
itself  bacteriocidal,  and  probably  this  effect  is  due  to  its  power 
of  increasing  digestion  and  stimulating  the  muscular  movements 
of  the  alimentary  canal,  thus  hurrying  the  food  in  its  passage 
through  this  region. 

Bile  serves  as  an  excretory  medium  for  certain  substances, 
among  them  cholesterol,  the  bile  pigments  and  other  materials. 

Bile  Pigments. — The  bile  contains  a  variety  of  colored  com- 
pounds, the  bile  pigments.  Bilirubin,  a  reddish  brown  pigment 
is  probably  the  mother  substance  of  most  of  the  others.  On 
oxidation  it  yields  biliverdin  (green),  bilicyanin,  and  other 
compounds.  On  reduction  it  yields  urobilin,  one  of  the  urinary 
pigments.     Tlie  bile  pigments  are  formed  by  the  breaking  down 


DIGESTION   IN    THE   INTESTINE  147 

of  hemoglobin  of  the  blood.  In  their  constitution  they  closely 
resemble  other  known  derivatives  of  hemoglobin.  They  contain 
no  iron.  There  has  been  much  discussion  as  to  where  the  trans- 
formation of  hemoglobin  into  bile  pigments  takes  place.  It  is 
probably  in  the  phagocytic  cells  of  the  liver,  which  are  believed 
to  engulf  the  red  corpuscles  and  destroy  them.  The  liver  cells 
then  finish  the  transformation. 

Bile  Salts. — The  bile  salts,  which  are  largely  responsible  for 
the  favorable  action  of  bile  in  digestion  and  absorption  of  fats, 
are  mainly  salts  of  glycocholic  and  taurocholic  acids,  two  con- 
jugated acids  made  up  of  cholic  acid  and  glycocoU  or  taurine 
respectively.  Cholic  acid  is  a  complex  substance  of  which  the 
formula  is  still  uncertain.  It  is  a  specific  product  of  liver  cells 
and  is  formed  nowhere  else  in  the  body.  Bile  salts  if  mixed  with 
a  little  sugar  and  brought  into  contact  with  concentrated 
sulphuric  acid  give  a  violet  color.  This  is  known  as  Petten- 
kofer's  test  for  bile  salts. 

Bile  contains  small  amounts  of  sodium  soaps  of  various  acids. 
Bile  from  the  bladder  also  contains  a  mucinous  substance  which 
is  perhaps  largely  a  phosphoprotein.  The  nature  of  this  sub- 
stance is  not  definitely  known. 

At  times  concretions  known  as  "gall  stones"  form  in  the  gall 
bladder.  They  are  com.posed  of  cholesterol,  inorganic  material 
or  bile  pigment  deposited  from  the  bile. 

Intestinal  Secretion 

The  third  of  the  important  digestive  fluids  poured  into  the 
intestine  is  called  the  intestinal  juice  or  succus  entericus. 
The  juice  secreted  into  the  duodenum  is  poured  out  by  small 
glands  in  the  mucous  membrane,  the  glands  of  Brun- 
ner.  Little  is  known  of  the  mechanism  by  which  these  glands 
are  made  to  secrete.  They  are  stimulated  to  activity  when 
the  acid  chyme  passes  the  pylorus.  The  amount  of  juice  secreted 
has  not  been  definitely  determined,  but  it  probably  is  large.  The 
juice  is  strongly  alkaline,  due  to  the  presence  of  carbonates,  and 


148  PHYSIOLOGICAL    CHEMISTRY 

thus  has  an  important  part  to  play  in  neutralizing  the  acid 
chyme.  This  is  by  no  means  the  only  role  of  the  intestinal  juice, 
however,  for  it  contains  substances  of  the  greatest  importance  in 
intestinal  digestion.  It  contains  enterokinase  which  greatly 
increases  the  action  of  pancreatic  juice  on  protein.  This  is 
usually  believed  to  be  due  to  activation  of  the  inactive  trypsino- 
gen,  by  conversion  into  trypsin.  Enterokinase  may  be  extracted 
from  the  intestinal  mucosa  from  almost  the  entire  length  of  the 
intestine  to  the  rectum,  but  it  is  present  in  largest  amount  in 
the  upper  regions  of  the  small  intestine.  Enterokinase  is  itself 
an  enzyme. 

Erepsin. — A  second  enzyme  found  in  the  intestinal  secretion 
is  erepsin.  This  enzyme  is  important,  for  it  is  responsible  for 
the  last  stage  in  the  digestion  of  proteins.  Erepsin  does  not  act 
on  proteins  themselves,  with  one  or  two  exceptions,  e.g.,  casein, 
but  on  the  intermediate  digestion  products,  proteoses,  peptones 
and  polypeptids,  which  it  reduces  to  amino  acids,  the  final  diges- 
tion products  of  the  proteiiis.  Erepsin  is  found  in  the  mucous 
membrane  of  the  intestinal  wall,  and  in  fact,  in  most  of  the  body 
tissues.     Erepsin  acts  best  in  weak  alkaline  solution. 

Other  Enzymes. — Three  enzymes  which  have  the  power  of 
splitting  disaccharides  also  are  present  in  the  intestinal  juice, 
maltase,  which  splits  maltose;  lactase,  which  splits  lactose,  and 
is  found  in  the  intestinal  juice  of  young  mammals  and  in  adults 
of  animals  which  take  milk  in  their  diet,  and  invertase,  which 
splits  cane  sugar. 

The  intestinal  juice  also  contains  a  nuclease,  which  splits  the 
nucleic  acids  of  the  nucleoproteins. 

Excretory  Function  of  Intestinal  Secretion. — Aside  from  the 
digestive  enzymes,  the  intestinal  juice  carries  various  substances 
into  the  intestine  which  are  thrown  out  of  the  blood  as  undesir- 
able, or  waste  materials.  It  thus  serves  in  the  capacity  of  an 
excretion  as  well  as  a  secretion. 

From  the  walls  of  the  large  intestine  a  secretion  is  poured 
into  the  intestinal  cavity.    This  contains  mucous  as  its  chief  con- 


DIGESTION   IN    THE    INTESTINE  149 

stitueiit,     and     appears     to     contain  no  enzymes  of  digestive 
importance. 

Bacterial  Action  in  the  Intestine. — The  intestine  shelters  and 
favors  the  growth  of  enormous  numbers  of  bacteria,  which  feed 
upon  the  substances  of  the  intestinal  contents.  From  these 
inaterials,  bacteria  produce  a  large  number  of  compounds,  some 
of  which  are  harmless,  but  some  extremely  toxic  to  the  body. 
The  toxic  materials  are  produced  mainly  from  the  digestion 
products  of  the  proteins,  the  amino  acids.  By  splitting  out 
COO  from  the  carboxyl  group  of  an  amino  acid  an  amine  is 
produced.  These  substances  are  known  as  ptomaines ;  many  are 
extremely  toxic.  An  example  of  this  process  is  the  formation  of 
ethyl  amine  from  alanine. 

CH3CH.  NH^COOH^CO^+CHgCH^NH^ 

From  tyrosine,  by  splitting  off  the  side  chain,  a  process  which 
goes  in  several  stages,  phenol  is  produced 

H  H 

C  C 

/\  /   \ 

HC       C  — CH2CHNH2COOH  HC       CH 

II  I  -^  II  I 

HO  C       CH  HO  C        CH 

\//  \   // 

C  c 

H  H 

Tyrosine  Phenol 

From  tryptophane  indol  and  skatol  are  produced. 

H  H 

C  C 

//   \  //   \ 

HC         C  —  C  —  CH^CHNHoCOOH      HC  C  —  C.CH3 

HC        C        C  HC  C        CH 

\/\/  \   /\/ 

C         N        ■  .  C  N 

H         H  H         H 

Tryptophane  Skatol 


150  PHYSIOLOGICAL   CHEMISTRY 

H 

C 

//   \ 
HC        C  —  CH 

HC        C        CH 

\   /\   / 

C        N 

H        H 

Indol 

From  cystin  and  cystein,  hydrogen  sulphide  and  niercaptans 
are  produced. 

Many  of  the  products  of  bacterial  action  are  not  harmful.  It 
is  even  possible  that  bacteria  may  in  some  ways  be  of  service  to 
the  organism,  as  in  attacking  and  breaking  down  indigestible 
substances,  and  producing  from  them  materials  which  can  be 
utilized  by  the  body.  In  putrefaction,  however,  many  harmful 
substances  are  produced,  as  above  stated,  and  are  responsible 
for  the  well-known  ill  effects  of  constipation.  The  material 
retained  in  the  intestine  putrefies,  the  products  are  absorbed 
into  the  blood,  and  give  rise  to  headaches  and  various  other 
symptoms  causing  discomfort. 

Putrefaction  is  greatly  favored  by  an  alkaline  reaction.  Also 
most  of  the  harmful  substances  resulting  from  putrefaction 
come  from  the  proteins.  Limiting  the  amount  of  protein  in  the 
diet  and  thoroughly  chewing  the  food  so  that  it  may  be  digested 
quickly  will  greatly  reduce  putrefaction  in  the  intestine. 

Feces. — In  every  mixed  meal  there  is  some  material  which  is 
indigestible  and  will  not  be  attacked  by  the  digestive  enzymes. 
Connective  tissue,  cellulose,  etc.,  are  examples  of  such  sub- 
stances. This  material  remains  in  the  intestine  and  forms  a 
part  of  the  solid  excreta,  or  feces.  In  addition,  the  feces  con- 
tain enormous  numbers  of  dead  bacteria.  Between  one  half  and 
one  fourth  of  the  solid  matter  of  the  feces  consists  of  dead  bac- 
teria. The  feces  also  contain  the  waste  material  of  the  cells  of 
the  intestinal  mucosa,  and  the  residues  of  the  digestive  secre- 
tions.   The  total  amount  of  solids  excreted  in  the  feces  per  day 


DIGESTION    IN    THE    INTESTINE  151 

averages  15-25  grams.  This  amount  may  vary  much  with  the 
nature  of  the  food.  The  feces  are  colored  chiefly  by  derivatives 
of  the  bile  pigments,  but  these  substances  themselves  do  not 
occur  here  ordinarily.  If  the  flow  of  bile  into  the  intestine  is 
inhibited,  the  feces  are  gray,  due  to  the  presence  of  fat  and  the 
absence  of  pigments.  Many  drugs  color  the  feces  black,  green, 
yellow,  etc. 


CHAPTER  X 
ABSORPTION 

General. — In  the  preceding  chapters  we  have  followed  the 
disintegration  of  the  various  foodstuffs  in  the  different  regions 
of  the  alimentary  canal.  By  the  combined  or  successive  action 
of  the  digestive  enzymes  and  other  agents  the  widely  varying 
carbohydrates,  fats  and  proteins  of  the  food  are  broken  down 
into  their  simple  building  stones,  monosaccharides,  fatty  acids 
and  glycerine,  and  amino  acids.  Thus  from  the  complex  foods 
a  mixture  of  simple  substances  is  obtained.  This  mixture  may 
vary  from  time  to  time  in  the  relative  amounts  of  the  different 
building  stones  it  contains,  but  the  kinds  of  material  it  contains 
are  fairly  uniform.  In  a  strict  sense  the  digestion  products  are 
not  yet  in  the  body  however.  The  alimentary  canal  is  nothing 
more  nor  less  than  a  tube  or  corridor  which  passes  through  the 
body.  Though  this  corridor  a  great  variety  of  materials  is 
made  to  pass.  These  materials  are  torn  to  pieces,  and  from  the 
resulting  simpler  substances  the  ever  ready  cells  of  the  walls  of 
the  alimentary  canal  select  and  take  up  certain  substances.  This 
process  is  called  absorption.  As  a  matter  of  fact  it  has  been 
much  debated  whether  absorption  is  merely  a  physical  diffusion 
of  material  through  the  walls  of  the  alimentary  tract,  or  whether 
it  involves  the  activities  of  the  cells  making  up  the  intestinal 
walls.    Undoubtedly  both  of  these  factors  come  into  play. 

Practically  no  absorption  takes  place  in  the  mouth,  and  but 
little  in  the  stomach.  There  is  some  evidence  that  salts,  mono- 
saccharides and  certain  other  substances  may  be  absorbed  to 
some  extent  from  the  stomach  if  they  are  present  in  high  con- 
centration, but  this  is  of  little  importance  normally. 

The  largest  part  of  the  absorption  takes  place  in  the  small 
intestine.     The  surface  area  of  the  intestine  is  greatly  increased 

152 


ABSORPTION  153 

by  finger-like  projections,  called  villi,  which  extend  into  the 
cavity  of  the  intestine.  The  villi  are  supplied  with  blood  ves- 
sels,— arteries,  capillaries  and  veins,  and  each  contains  also  a 
lacteal,  or  lymph  vessel.  The  walls  of  the  villi  are  very  thin, 
so  that  only  a  thin  membrane  separates  the  food  in  the  intestinal 
contents-  from  the  capillaries  and  lacteals.  The  cells  of  this 
membrane  are  alive,  and  whereas  simple  processes  of  diffusion 
probably  account  for  a  portion  of  the  absorption  of  food,  the 
living  cells  of  the  intestinal  wall  undoubtedly  influence  the 
process  by  taking  up  particular  substances  and  passing  them,  or 
products  made  from  them,  on  into  the  blood  in  the  capillaries 
or  into  the  lymph  in  the  lacteals. 

Absorption  of  Proteins. — Proteins  are  absorbed  mainly  in  the 
form  of  amino  acids.  A  portion  of  these  are  passed  directly  into 
the  blood  of  the  capillaries  and  thus  enter  the  blood  stream  by 
which  they  are  carried  to  the  different  tissues  and  cells.  Pos- 
sibly a  portion  of  the  amino  acids  are  deaminized, — that  is,  lose 
their  amino  groups  in  their  passage  through  the  intestinal  wall. 
Little  is  known  of  the  further  fate  of  the  residue  left  after 
splitting  off  the  amino  group.  Perhaps  it  is  burned  by  the  cells 
of  the  body  as  fuel,  or  used  to  build  up  new  substances  in  the 
cells.  Amino  acids  have  been  obtained  from  the  blood  or  shown 
to  be  there  by  various  methods,  so  that  there  is  no  longer  doubt 
that  this  is  the  form  in  which  the  proteins  of  the  food  reach  the 
cells.  This  subject  was  long  in  dispute,  for  the  amount  of  amino 
acids  in  the  blood  at  any  one  time  is  so  small  that  only  recently 
has  it  been  possible  to  prove  that  they  are  there  at  all. 

Carbohydrate  Absorption. — The  monosaccharides  glucose, 
levulose  and  galactose  produced  by  the  digestion  of  the  carbohy- 
drates are  believed  to  pass  into  the  blood  of  the  capillaries  and 
thence  into  the  blood  stream.  Glucose  is  found  regularly  in  the 
blood  to  the  extent  of  0.2-0.4%.  The  liver  takes  up  monosac- 
charides and  builds  them  into  glycogen,  which  serves  as  a  reserve 
store  of  fuel.     Glycogen  is  stored  also  in  the  muscles. 

At  times,  if  excessive  amounts  of  maltose,  lactose,  or  cane 
sugar  are  taken,  these  sugars  may  get  into  the  blood.    The  mal- 


154  PHYSIOLOGICAL    CHEMISTRY 

tose  will  be  split  and  utilized  at  least  in  part,  as  the  blood  eon- 
tains  a  maltase.  Blood  appears  to  contain  little  or  no  lactase 
or  invertase,  however,  so  that  lactose  or  cane  sugar  which  get 
into  the  blood  are  excreted  into  the  duodenum,  or  in  the  urine. 

Absorption  of  Fats.— Fats  are  absorbed  mainly  in  the  form 
of  fatty  acids  and  soaps.  These  are  recombined  with  glycerine 
in  the  cells  of  the  intestinal  wall,  and  pass  into  the  lacteals  as 
neutral  fat. 

In  the  absorption  of  fats  the  bile  plays  an  important  role. 
If  bile  is  excluded  from  the  intestine,  the  absorption  of  fats  is 
greatly  diminished.  Large  amounts  of  fatty  acids,  soaps,  and 
some  fat  appear  in  the  feces,  indicating  •  that  the  fatty  acids 
and  soaps  are  absorbed  very  slightly  in  the  absence  of  bile. 
Bile  aids  in  the  absorption  process  by  its  power  of  dissolving 
fatty  acids  and  soaps.  These  soaps  and  fatty  acids  dissolved 
by  the  bile  pass  into  the  cells  of  the  intestinal  wall.  Glycerine 
also  is  absorbed,  and  from  this  and  the  fatty  acids,  neutral  fat 
is  constructed.  This  passes  for  the  most  part  into  the  lacteals, 
thence  along  the  lymphatics  and  into  the  thoracic  duct  whence 
it  is  poured  into  the  blood  stream  at  the  junction  of  the  jugular 
and  subclavian  veins:  The  bile  salts  which  have  entered  the 
blood  with  the  fatty  acids  are  picked  out  of  the  blood  by  the 
liver  and  returned  to  the  bile. 

During  digestion,  Avhite  corpuscles  are  known  to  gather  in 
large  numbers  in  the  neighborhood  of  the  intestine.  Evidently 
they  are  concerned  in  some  way  in  taking  care  of  the  absorbed 
foodstuffs,  but  their  role  is  as  yet  a  matter  of  uncertainty. 

Most  of  the  absorption  of  digestive  products  takes  place  in  the 
small  intestine,  so  that  little  useful  material  is  left  by  the  time 
the  digested  food  reaches  the  large  intestine.  In  this  region, 
however,  there  is  a  large  absorption  of  water  so  that  this  valu- 
able liquid  is  not  wasted  by  excretion  with  the  feces. 


CHAPTER  XI 
URINE 

General. — The  body  is  by  no  means  a  permanent  structure 
if  considered  from  the  standpoint  of  the  individual  molecules  of 
which  it  is  composed.  The  tissues  are  constantlj^  wearing  out 
and  being  rebuilt  in  the  process  of  wear  and  tear.  In  this 
process  waste  products  are  formed.  For  the  maintenance  of 
body  temperature  and  the  performance  of  mechanical  work,  sub- 
stances are  constantly  being  ''burned"  or  oxidized  in  the  body, 
both  substances  from  the  food,  or  from  the  organic  reserves  of 
the  tissues  themselves.  Here  also  waste  products  are  produced. 
The  body  must  dispose  of  waste  material.  This  is  accomplished 
by  way  of  the  various  excretions, — through  skin,  lungs  and 
kidneys,  and  in  the  feces. 

The  skin  excretes  water  and  salts,  and  about  a  gram  of  nitro- 
gen per  day  in  various  compounds.  From  the  lungs  much  water 
and  carbon  dioxide  are  given  off.  The  feces  contain  mainly  undi- 
gested food  residues,  the  dead  bodies  of  intestinal  bacteria  and 
substances  from  the  digestive  secretions,  but  also  some  other 
materials  which  are  excreted  into  the  intestine.  They  contain 
1-2  grams  of  nitrogen  per  day. 

Most  of  the  nitrogen  excreted  is  given  off  in  the  urine  how- 
ever, and  hence  this  excretion  attracts  especial  interest ;  a  study 
of  the  amounts  and  variations  in  the  nitrogen  elimination  often 
will  give  valuable  information  as  to  what  is  going  on  in  the  body 
itself.  This  is  not  confined  entirely  to  nitrogen  compounds,  for 
the  urine  contains  various  other  substances  of  interest.  Occa- 
sionally also,  abnormal  constituents  appear,  and  by  their  pres- 
ence or  amount  give  information  which  is  of  great  value  to  the 
physician.  The  study  of  the  urine  is  thus  of  primary  impor- 
tance. 

155 


156  PHYSIOLOGICAL   CHEMISTRY 

Physical  Properties 

Volume. — The  excretion  of  the  various  urine  constituents 
varies  considerably  during  the  course  of  the  24  hour  day. 
Except  in  the  case  of  some  pathological  constituents,  the  mere 
presence  of  which  is  an  indication  of  an  abnormal  condition,  it 
is  customary  to  make  analysis  of,  and  report  the  amounts  of  the 
substances  found  in  the  urine  voided  during  a  complete  24 
hour  period.  Such  a  24  hour  specimen  is  most  conveniently  col- 
lected by  discarding  the  first  voiding  in  the  morning,  then  col- 
lecting all  urine  voided  during  the  day,  and  the  first  voiding  of 
the  folloAving  morning.  The  specimen  should  be  preserved  from 
spoiling  by  adding  5-10  c.c.  of  a  5%  thymol  solution  in  chloro- 
form. 

The  volume  of  such  a  sample  varies  through  fairly  wide 
limits.  It  is  determined  by  the  balance  between  the  amount  of 
water  taken,  and  that  excreted  in  other  ways.  Thus,  loss  by 
excessive  sweating,  by  diarrhea,  or  vomiting,  or  in  fever  where 
evaporation  from  the  skin  is  increased  will  cause  a  fall  in  the 
urine  volume.  On  the  other  hand,  drinking  much  water,  or 
prevention  of  loss  through  the  skin  as  on  humid  days  when 
evaporation  is  low,  will  tend  to  increase  the  total  volume  of  the 
urine.  An  average  figure  for  adult  men  is  1,000-1,200  c.c.  per 
day,  in  women  somewhat  less,  but  the  volume  from  a  perfectly 
healthy  subject  may  be  much  lower  (400-500  c.c.  or  less)  or  much 
higher  (2  to  three  liters  or  more).  Children  excrete  less  urine 
than  adults,  600-700  c.c.  being  an  average  amount  between  the 
ages  of  3  and  7  or  8  years.  A  new  born  infant  excreted  17  c.c. 
the  first  day,  and  on  the  6th  day,  206  c.c.  Vegetarians  usually 
excrete  a  small  volume. 

In  pathological  conditions  such  as  fevers,  or  in  kidney  affec- 
tions, the  volumes  may  greatly  diminish  (oliguria),  or  no  urine 
may  be  excreted  at  all.  Such  a  condition  is  of  course  extreme. 
In  the  various  forms  of  diabetes  the  volume  may  be  very  large ; 
even  10  liters  per  day  and  greater  volumes  have  been  reported. 
Such  a  condition  is  known  as  polyuria. 


URINE  157 

Color,  Transparency. — The  color  of  normal  urine  ranges  from 
a  pale  straw  color  to  a  dark  brown.  Ordinarily  it  is  amber.  The 
color  is  due  to  the  presence  of  pigments, — urochrome  and  uro- 
bilin being  of  greatest  importance.  Uroerythrin  may  give  normal 
urine  or  its  sediments  a  reddish  color.  Dilute  urines  of  large 
volume  usually  are  pale  in  color,  concentrated  urines  usually 
darker.  The  reaction  of  the  specimen  also  affects  the  color,  as 
acid  urines  are  usually  darker,  alkaline  urines  lighter. 

Various  pathological  colorings  may  occur, — thus  blood  pig- 
ment or  its  derivatives  may  cause  a  red  or  brown ;  bile  pigments 
give  the  urine  a  dark  brown,  greenish,  or  greenish  black  color.  In 
a  condition  known  as  alcaptonuria  the  urine  turns  dark  on  stand- 
ing. It  should  be  borne  in  mind  that  various  drugs  will  color 
the  urine,  such  as  senna  (yellow),  tar  preparations,  salol,  etc. 
(brown).  Also  madder,  beets,  or  the  analine  dyes  used  in  cheap 
candies  may  result  in  abnormal  colorations. 

The  transparency  of  normal  urine  varies  greatly.  Fresh  acid 
urine  usually  is  clear  or  fairly  so.  If  concentrated,  it  may  be 
cloudy  from  a  precipitate  of  urates  or  uric  acid.  This  precipi- 
tate will  dissoh'e  on  warming.  On  standing,  a  slight  sediment 
usually  settles  out.  This  consists  of  various  cells  or  cell  debris, 
urates,  uric  acid  and  some  other  substances. 

If  the  specimen  is  alkaline,  it  usually  is  cloudy  from  precipi- 
tated phosphates  or  carbonates  of  the  alkaline  earths.  This 
cloudiness  does  not  disappear  on  warming,  but  does  on  acidi- 
fying. 

Pathological  urine  may  be  cloudy  from  mucin,  epithelial  cells, 
pus,  blood,  bacteria,  etc. 

To  clear  a  cloudy  specimen  filtering  often  is  sufficient.  If 
the  urine  is  alkaline,  the  addition  of  acetic  acid  may  cause 
clearing. 

Decolorizing  may  be  brought  about  by  adding  charcoal,  or 
permanganate  or  in  other  ways.  The  method  chosen  should  not 
interfere  with  the  analysis  to  be  undertaken  however. 

Albumin  may  be  removed  by  the  careful  addition  of  acetic 
acid  and  boiling.     Care  should  be  taken    (trial  and  error)    to 


158  PHYSIOLOGICAL    CHEMISTRY 

add  just  the  right  amount  of  acid,  as  excess  or  a  deficient  amount 
will  result  in  incomplete  precipitation  of  the  protein. 

Consistency,  Odor,  Taste. — The  urine  usually  is  thin  and 
watery.  It  foams  on  shaking  but  the  foam  quickly  disappears. 
If  the  urine  is  albuminous  the  foam  may  persist.  If  it  contains 
much  mucin  or  pus,  the  urine  may  be  thickish  in  character. 

The  odor  of  fresh  urine  somewhat  resembles  that  of  a  meat 
broth.  It  is  quite  characteristic.  Little  is  known  of  the  nature 
of  the  substances  responsible  for  the  odor.  Eecently  a  substance 
"urinod"  has  been  reported.  When  urine  is  allowed  to  stand 
without  a  preservative,  it  quickly  acquires  a  sharp  ammoniacal 
odor.  Decomposition  has  taken  place.  With  a  little  practice, 
the  analyst  is  able  to  detect  w^hen  a  urine  has  ' '  spoiled, ' '  and  be- 
come unsuitable  for  analysis.  Albumin  or  pus  urine,  especially 
if  old,  often  has  a  -putrid  odor.  Ingested  drugs  or  foods  may 
cause  an  unusual  odor, — thus  asparagus,  or  onions  give  the  urine 
a  disagreeable  odor.  After  taking  menthol,  an  odor  of  pepper- 
mint is  observed. 

The  taste  of  normal  urine  usually  is  somewhat  salty  due  to 
NaCl.    Diabetic  urine  may  taste  sAveet,  from  the  sugar  present. 

Specific  Gravity. — Total  Solids. — The  specific  gravity  of  nor- 
mal urine  obviously  depends  upon  the  relation  between  the 
amount  of  total  solids,  and  the  volume  of  the  urine.  An  average 
specimen  will  have  a  specific  gravity  of  from  1.017-1.020,  but  it 
may  fall  to  1.010  or  lower  if  the  volume  is  large,  or  rise  to  1.030 
or  higher  if  the  volume  is  low  or  the  total  solids  high.  In  new 
born  infants  the  specific  gravity  is  low,  about  1.005-1.007. 

In  pathological  conditions  the  volume  and  specific  gravity 
may  not  vary  inversely.  Thus  in  diabetes  mellitus  a  large  vol- 
ume is  accompanied  by  a  fairly  high  specific  gravity,  on  account 
of  the  sugar  present.  In  cases  of  albuminuria  the  volume  may 
be  low  and  the  specific  gravity  low  also. 

Specific  gravity  usually  is  estimated  by  means  of  an  areom- 
eter or  urinometer.     (See  laboratory  directions  for  urine.) 

The  total  solids  excreted  in  the  course  of  a  day  will  vary 
considerably.    Sixty  grams  is  an  average  figure.    The  amount  de- 


URINE  159 

pends  largely  upon  the  amounts  of  sodium  chloride  and  pro- 
tein eaten,  for  xNTaC'l  and  urea,  the  principal  end  product  of 
protein  metabolism  in  the  body,  make  up  the  largest  part  of  the 
total  solids.  (For  the  estimation  of  total  solids  see  the  labora- 
toi-y  directions.) 

Optical  Activity,  Reducing  Power,  Fermentation,  Etc. — 
Normal  urine  is  slightly  levorotatory,  since  it  contains  minute 
traces  of  protein  and  of  conjugated  glucuronates,  both  of  which 
rotate  strongly  to  the  left.  It  contains  also  minute  traces  of 
glucose,  which  is  dextrorotatory,  but  the  amount  is  too  small  to 
counteract  the  levorotatory  substances. 

Pathological  urine  may  show  strong  rotation.  If  dextrose  is 
present  the  rotation  will  be  to  the  right.  If  protein  or  levulose 
are  present  in  quantity,  the  rotation  mil  be  to  the  left,  as  is  also 
the  case  if  the  urine  contains  large  amounts  of  conjugated  glu- 
curonates, as  it  does  after  taking  camphor,  chloral  hydrate, 
menthol  and  various  other  drugs. 

Normal  urine  has  a  slight  reducing  power,  due  to  its  content 
of  traces  of  sugar,  of  conjugated  glucuronates,  uric  acid  and 
other  substances.  The  reduction  is  not  sufficient  to  interfere  in 
the  ordinary  Fehling  test  when  small  amounts  of  urine  are  used 
however,  so  that  this  test,  as  performed  with  normal  urine  is 
negative.  In  pathological  urine,  particularly  carbohydrate 
urine,  the  reduction  may  be  extensive,  and  is  made  use  of  to 
detect  sugar. 

On  standing,  urine  may  ferment  in  a  variety  of  w^ays  as  the 
result  of  the  activities  of  microorganisms.  Ammoniacal  fer- 
mentation is  the  most  common.  Micrococcus  urese  and  B.  urese 
decompose  the  urea,  forming  ammonia.  The  urine  becomes  alka- 
line, its  color  changes,  and  phosphates,  etc.,  are  precipitated. 
Dilute  urine  usually  ferments  quicker  than  a  concentrated 
specimen.  A  strong  acid  reaction  retards  the  process.  Other 
types  of  fermentation  also  occur. 

Pathological  urines  usually  ferment  quicker  than  normal 
specimens,  as  they  furnish  a  good  medium  for  the  development 
of  microorganisms.     Occasionally,   fermentation   occurs  in  the 


160  PHYSIOLOGICAL    CHEMISTRY 

bladder  before  the  urine  is  voided.     Various  gases  may  be  pro- 
duced, the  condition  being  known  as  pneumaturia. 

If  injected  into  the  blood  stream,  urine  has  a  toxic  effect.  This 
is  due  in  part  perhaps  to  certain  alkaloidal  substances  present 
in  small  amounts,  but  also  to  disturbances  in  osmotic  equilib- 
rium. 

Spectroscopic  examination  of  the  urine  is  sometimes  valuable 
in  the  detection  of  blood  or  bile  pigments. 

Reaction. — The  urine  of  a  normal  healthy  individual  may 
vary  considerably  in  chemical  reaction, — thus  it  may  be  acid, 
neutral  or  alkaline.  The  reaction  depends  primarily  on  the 
nature  of  the  diet.  On  a  meat  (protein)  diet,  the  urine  is  acid, 
on  a  vegetable  (non-protein)  diet  the  urine  may  be  alkaline. 
Thus  the  urine  of  carnivora  is  acid,  that  of  herbivora  alkaline. 
If  either  class  of  animal  is  forced  to  eat  the  other  class  of 
material,  the  reaction  of  the  urine  changes  accordingly.  In 
starA^ation,  the  urine  is  acid,  since  an  animal  thus  becomes  car- 
nivorous, living  upon  its  own  tissues.  At  the  beginning  of  gastric 
digestion,  the  urine  usually  is  alkaline,  due  to  the  abstraction 
of  available  H-ions  from  the  blood  to  form  the  gastric  HCl. 
Profuse  sweating  also  may  lower  the  acidity,  since  acid  is  carried 
out  through  the  skin. 

The  effect  of  variations  in  diet  upon  the  reaction  of  the  urine 
is  due  to  the  products  formed  in  the  breaking  down  of  food 
constituents.  Proteins  contain  sulphur  and  phosphorus,  which 
are  transformed  into  sulphuric  and  phosphoric  acids,  and 
excreted  largely  in  the  form  of  acid  salts  and  probably  to  a 
slight  extent  as  the  free  acids.  These  two  factors  are  mainly 
responsible  for  the  acid  reaction  of  urine,  although  there  also 
are  small  amounts  of  various  organic  acids.  The  organic  acids 
in  vegetable  products,  on  the  other  hand,  are  oxidized  to  CO2 
in  the  body,  a  portion  of  which  is  excreted  in  the  urine  as  car- 
bonate. The  hydrolysis  of  these  salts  is  responsible  for  an  alka- 
line reaction  of  urine.  Ingesting  organic  acids  such  as  citric, 
malic,  etc.,  which  are  burned  to  carbonates,  actually  increases 


URINE 


161 


the  alkalinity  of  the  urine,  thus  bearing  out  the  above  con- 
elusions. 

As  might  be  (Expected,  the  total  titratable  acidity  of  the  urine 
varies  greatly.  An  average  24  hour  specimen  on  a  mixed  diet 
will  require  150-400  c.c.  N/10  alkali  to  neutralize  it,  using 
phenolphthalein  as  indicator.  These  figures  may  be  exceeded  in 
either  direction. 

There  appear  to  be  no  consistent  and  characteristic  variations 
in  acidity  in  disease. 

The  hydrogen-ion  concentration  of  urine  may  be  determined 
by  the  gas  chain  method,  or  by  the  use  of  a  series  of  indicators 
such  as  that  worked  out  by  Henderson.  The  average  value  is 
NXlO-^  but  it  ranges  between  NXlO''-'^  and  NXlO"'*. 

/NH2 
Urea. — 0  =  C  .    By  far  the  largest  part  of  the  nitrogen 

XNH, 

excreted  by  the  body  is  in  the  form  of  urea.  Other  nitrogenous 
substances  are  uric  acid,  ammonia,  creatinine  and  creatine,  hip- 
puric  acid,  allantoin,  amino  acids  and  a  variety  of  other  sub- 
stances. Of  the  total  nitrogen  contained  in  all  these  substances, 
urea  contains  on  an  average  85-90%.  On  a  high  protein  diet 
this  percentage  may  increase,  and  on  a  low  protein  diet  it  may 
fall  even  as  low  as  60%.  Also  the  total  amount  of  urea  varies 
with  the  amount  of  protein  in  the  food.  A  variation  of  from 
8  or  10  grams  to  30  grams  per  day  will  cover  most  cases. 

Urea  is  a  colorless,  odorless  compound  which  crystallizes  in 
long  needles.  It  is  almost  tasteless,  but  produces  a  sensation  of 
coolness  when  placed  on  the  tongue.  It  is  readily  soluble  in 
water  and  alcohol  but  insoluble  in  ether.  With  nitric  or  oxalic 
acid,  it  forms  urea  nitrate  or  oxalate  respectively,  one  molecule 
of  nitric  uniting  with  one  of  urea,  one  of  oxalic  with  two  of 
urea.  These  salts  crystallize  easily  and  are  useful  in  the  isola- 
tion and  purification  of  urea. 

On  heating  dry  urea,  it  decomposes,  forming  biuret,  the  sub- 
stance for  which  the  biuret  test  was  named,  cyanuric  acid  and 
other  substances. 


162  PHYSIOLOGICAL    CHEMISTRY 


NH2 

/ 

NH2 

c=o 

/ 

\ 

2     C  =  0 

-^           NH 

\ 

/ 

NH2 

c=o 

\ 

NH2 

Biuret, 

Urea  is  decomposed  by  nitrous  acid,  and  by  hypobromite ;  the 
nitrogen  is  liberated  as  the  gas,  and  may  be  measured.  Various 
methods  for  the  quantitative  estimation  of  urea  depend  upon 
these  reactions.  If  heated  to  153°  C.  urea  gives  up  its  nitrogen 
as  ammonia.  This  method  also  has  been  used  in  some  of  the  most 
successful  quantitative  urea  methods.  An  enzyme  urease  which 
is  found  in  the  soy  bean,  and  elsewhere,  has  the  property  of  de- 
composing urea,  but  no  other  nitrogenous  constituent  of  the 
urine.  The  urea  is  converted  into  ammonium  carbonate  from 
which  the  ammonia  easily  may  be  liberated.  This  is  the  basis  for 
the  method  of  urea  determination  in  general  use  at  present. 

The  original  source  of  urinary  urea  is  the  proteins  of  food  and 
tissues.  Proteins  are  made  up  of  amino  acids.  In  digestion  they 
are  split  up  into  these  compounds.  Where  and  how  is  the  amino 
acid  nitrogen  converted  into  urea?  This  is  a  question  which 
has  taken  much  labor  to  solve.  A  portion  of  the  amino  acids  are 
deaminized  by  bacteria  in  the  intestine,  a  portion  possibly  in 
their  passage  through  the  cells  of  the  intestinal  wall.  The  amino 
group  is  converted  into  ammonia  which  circulates  in  the  blood  as 
ammonium  carbonate.  It  has  been  shown  that  an  excised  liver 
if  perfused  with  blood  containing  ammonium  carbonate  will  con- 
vert this  substance  into  urea,  but  most  of  the  amino  acids  pass 
into  the  blood  after  absorption.  Amino  acids  undoubtedly  are 
deaminized  in  the  liver  itself,  and  also  in  various  other  tissues,  so 
that  the  supply  of  ammonium  carbonate  may  come  from  very 
generally  distributed  regions.  An  attempt  has  been  made  to 
demonstrate  that  the  manufacture  of  urea  occurs  only  in  the 


URINE  163 

liver,  but  evidence  does  not  bear  out  this  assumption.  Probably 
urea  formation  is  a  function  of  all  cells  of  the  body,  but  is  par- 
ticularly prominent  in  liver  tissues.  The  liver  cannot  be  removed 
from  the  body  of  a  mammal  without  causing  the  death  of  the  ani- 
mal in  a  few  hours.  The  liver  may  be  shunted  out  of  the  circu- 
lation, however,  by  an  operative  procedure  known  as  Eck's  fis- 
tula. In  an  Eck's  fistula,  the  portal  vein  is  joined  to  the  inferior 
vena  cava.  The  portal  vein  beyond  the  fistula  is  then  ligated. 
The  blood  from  the  intestine  now  no  longer  passes  to  the  liver,  but 
into  the  vena  cava  and  thence  to  the  heart.  The  liver  still  receives 
some  blood  by  way  of  the  hepatic  artery  but  most  of  the  blood 
does  not  pass  through  the  liver.  Under  these  circumstances,  the 
amount  of  urea  decreases  greatly,  with  a  corresponding  increase 
in  ammonia.  It  also  has  been  observed  that  in  various  acute  dis- 
eases of  the  liver  or  in  injury  to  the  liver  cells  after  poisoning 
with  certain  substances,  a  fall  in  urinary  urea  results.  This  and 
other  evidence  points  to  the  conclusion  that  most  of  the  urea  is 
formed  in  the  liver,  but  that  it  also  is  formed  elsewhere,  perhaps 
generally  in  the  body. 

A  small  proportion  of  the  urinary  urea  may  arise  from  the 
direct  hydrolysis  of  one  of  the  amino  acid  constituents  of  the  pro- 
teins, viz.  arginine.  Inspection  of  the  formula  for  this  compound 
will  demonstrate  the  ease  of  this  process  from  a  chemical  stand- 
point. There  probably  also  are  various  other  minor  sources  of 
urea. 

Urea  has  a  distinct  physiologic  action,  acting  as  a  diuretic. 
Increase  of  urea  in  the  blood  increases  the  flow  of  urine.  This 
is  in  harmony  with  the  fact  that  on  high  protein  diet,  the  volume 
of  the  urine  also  is  high. 

Uric  Acid  and  Other  Purine  Derivatives.— Uric  acid  occurs 
in  the  urine  in  amounts  ranging  from  about  0.3  to  1.2  grams  per 
day.  It  makes  up  from  5-10%  of  the  total  nitrogen  of  the  urine. 
Notwithstanding  its  small  amount,  uric  acid  is  of  great  interest, 
and  long  has  attracted  widespread  attention  because  it  occurs  as 
a  urinary  sediment.     On  account  of  its  insoluble  character,  it  is 


164  PHYSIOLOGICAL   CHEMISTRY 

deposited  in  the  joints  in  gout  and  arthritis,  and  shows  other 
interesting  variations  in  disease  and  on  varying  diets. 

In  birds  and  reptiles  uric  acid  is  the  chief  nitrogenous  excre- 
tion. The  urine  of  these  animals  is  semisolid,  since  it  contains 
large  amounts  of  uric  acid  crystals. 

Uric  acid  is  2,  6,  8  trioxypui-ine  and  has  the  following  formula : 

HN  —  C  =  0 

I      ! 

0==C      C  — NH 
\ 

c==o 

/ 

HN  — C  — NH 

Uric  Acid. 

It  is  extremely  insoluble  and  frequently  precipitates  from  the 
urine  as  a  crystalline  sediment  which  usually  is  highly  colored 
by  other  substances.  The  crystal  form  varies,  but  is  frequently 
whetstone  or  gunboat  shape.  It  dissolves  fairly  readily  in  alka- 
lies, in  boiling  glycerol,  and  in  concentrated  sulphuric  acid  on 
slight  warming  without  decomposition.  It  forms  salts,  those  of 
sodium  and  amonium  often  being  present  in  urine  sediment.  Uric 
acid  is  easily  decomposed  in  alkaline  solution,  and  yields  various 
products  such  as  urea,  dialuric  acid,  etc.  It  reduces  Fehling's 
solution,  and  is  readily  oxidized  by  alkaline  permanganate  and 
other  oxidizing  agents.  Alloxan,  allantoin,  oxalic  and  carbonic 
acids  are  among  the  oxidation  products  of  uric  acid. 

If  a  crystal  of  uric  acid  is  moistened  with  concentrated  HNO3 
and  evaporated  to  dryness  on  the  water  bath  a  reddish  spot  re- 
mains, which  becomes  a  deeper  red  on  the  addition  of  ammonia. 
This  is  known  as  the  murexide  test.  Other  purines  (xanthine 
and  guanine)  give  this  test,  but  may  be  distinguished  from  uric 
acid  by  the  fact  that  the  red  color  persists  on  warming,  whereas 
that  from  uric  acid  disappears  on  warming.  The  red  substance 
from  uric  acid  is  ammonium  purpurate. 

The  source  of  the  uric  acid  was  long  a  matter  of  uncertainty. 
We  now  know,  however,  that  it  is  derived  from  the  purine  por- 


URINE  165 

tion  of  nucleic  acid.  Thus  the  nucleoproteins  are  the  ultimate 
source.  This  fact  increases  interest  in  uric  acid,  since  evidently  it 
is  a  product  of  the  breaking  down  or  catabolism  of  nuclear  ma- 
terial. The  purine  bases,  adenine  and  guanine,  are  constituents  of 
nucleic  acid.  In  the  body  these  compounds  are  oxidized  to 
h^TJoxanthine  and  xanthine,  and  these  latter  substances  to  uric 
acid.  These  purines  may  come  from  the  breaking  down  of  nu- 
clear material  in  the  food,  or  in  the  tissues  themselves.  This 
fact  may  be  demonstrated,  for  if  glandular  material  such  as 
sweet  breads  (pancreas)  is  fed,  there  is  an  increase  in  the  uric 
acid  of  the  urine.  Since  this  uric  acid  has  an  origin  outside  the 
tissues,  and  comes  from  material  which  probably  is  at  no  time  a 
constituent  of  the  cells,  it  is  called  exogenous  uric  acid,  i.  e.,  com- 
ing from  without.  It  is  interesting  that  all  of  the  ingested 
purines  are  not  recovered  in  the  urine  as  uric  acid.  Evidently  a 
portion  either  is  not  absorbed,  or  is  destroyed  in  the  body.  If 
an  animal  is  kept  upon  a  purine  free  diet,  uric  acid  does  not  dis- 
appear from  the  urine;  from  0.3-0.5  grams  a  day  still  is 
excreted.  This  must  come  from  the  nuclear  material  of  the  tissues 
themselves,  and  hence  is  called  endogenous  uric  acid,  i.e.,  coming 
from  within.  There  is  a  possibility  that  at  last  a  portion  of 
this  uric  acid  comes  from  the  nuclear  material  of  dead  bacteria 
in  the  intestine,  but  it  is  generally  considered  to  arise  mainly  in 
the  catabolism  of  the  tissue  nucleins. 

The  transformation  of  the  purines,  adenine  and  guanine,  into 
uric  acid  involves  several  steps.  Quite  recently  this  process  has 
been  carefully  studied,  and  enzymes  have  been  found  in  vari- 
ous tissues  which  possess  the  property  of  carrying  out  all  the 
intermediate  stages  of  the  process. 

In  most  mammals  an  enzyme  uricase  occui^s  which  has  the 
power  of  destroying  uric  acid, — allantoin  being  one  of  the  chief 
products.  This  enzyme  has  not  been  found  in  man,  however, 
and  it  is  probable  that  man  has  lost  the  power  of  destroying  uric 
acid.  To  be  sure,  if  uric  acid  is  given  to  a  human  being  by 
mouth,  only  a  small  portion  of  it  appears  in  the  urine.  This 
fact  is  still  unexplained.  Perhaps  intestinal  bacteria  are  respon- 
sible for  its  destruction. 


166  PHYSIOLOGICAL    CHEMISTRY 

The  question  has  been  raised,  can  the  body  build  up  its  own 
purines  (and  from  these  its  nucleoproteins)  from  non-purine 
substances?  This  question  is  as  yet  unanswered.  In  birds  and 
reptiles  the  synthesis  of  uric  acid  is  a  well  known  fact.  In 
invertebrates  also  this  synthesis  takes  place.  It  would  be  curious 
if  only  mammals  lacked  this  power.  We  know  in  fact  that  in 
very  young  mammals  living  on  milk,  which  contains  only  traces 
of  purine,  large  amounts  of  nuclear  material  are  built  up, 
apparently  from  non-purine  sources.  But  the  question  is  still 
unsettled,  and  awaits  further  evidence. 

The  variations  in  the  amount  of  uric  acid  in  disease  are  in- 
fluenced by  the  amount  of  destruction  of  nuclear  material  in  the 
body.  During  recovery  from  pneumonia,  when  the  transudates 
containing  large  numbers  of  leucocytes  are  being  reabsorbed,  in 
leucemia  where  the  number  of  leucocytes  in  the  blood  is  great- 
ly increased,  after  severe  burns  which  have  caused  the  disin- 
tegration of  much  tissue  and  in  pregnancy  when  there  is  in- 
creased nuclear  metabolism,  an  increase  in  uric  acid  is  ob- 
served. In  gout  and  arthritis  uric  acid  is  deposited  in  the  joints 
causing  much  pain  and  inconvenience.  It  is  a  debated  question, 
however,  as  to  whether  this  is  the  cause  or  the  effect  of  the  dis- 
ease. 

Other  purines  occur  in  the  urine  in  small  quantities.  Also 
eaffein,  theobromine  and  other  members  of  the  group  which 
occur  in  coffee,  cocoa,  etc.,  may  be  present.  The  quantitative  esti- 
mation of  uric  acid  is  described  in  the  laboratory  directions. 

Hippuric  Acid, — 

0 

II 
C  —  C  — NH  — CH2  COOH 

//\ 
HC         CH 

I  II 

HC         CH 

\/ 
C 
H 


UEINE 


167 


Hippuric  acid  is  found  in  large  amounts  in  the  urine  of  her- 
bivora.  Less  than  a  gram  a  day  usually  is  present  in  the  urine 
of  man.  It  is  interesting,  for  it  is  a  substance  formed  by  the 
union  of  benzoic  acid  and  glycocoll.  The  former  may  be  formed 
in  the  body,  or  may  be  ingested  with  the  food,  as  it  occurs  in 
various  fruits  and  vegetables,  particularly  in  cranberries.  The 
formation  of  hippuric  acid  apparently  is  a  protective  measure, 
as  benzoic  acid  is  considerably  more  toxic  than  hippuric.  The 
benzoic  is  conjugated  with  glycocoll,  which  comes  in  part  from 
the  destruction  of  protein  tissue  or  food,  but  also  may  be  synthe- 
sized by  the  body.  The  hippuric  acid  is  then  excreted,  and  the 
more  dangerous  benzoic  acid  thus  removed. 

Ammonia. — The  urine  always  contains  small  amounts  of  am- 
monia, usually  less  than  a  gram  a  day.  Ammonia  is  used  by  the 
body  to  neutralize  acids  which  are  not  oxidized  and  destroyed 
by  the  body.  The  taking  of  a  mineral  acid  thus  will  cause  an  in- 
crease in  urinary  ammonia.  This  occurs  also  in  diabetes,  where 
aceto  acetic  acid  and  ^-oxybutyric  acid  are  produced  and  not 
further  oxidized  in  the  organism.  They  are  excreted  in  the  form 
chiefly  of  their  ammonium  salts.  The  source  of  the  ammonia  is 
largely  the  split-off  amino  groups  liberated  in  the  destruction  of 
amino  acids  when  proteins  are  destroyed.  Most  of  this  ammonia 
is  built  into  urea,  but  a  portion  is  excreted  as  such.  For  the 
quantitative  estimation  of  ammonia,  see  the  laboratory  direc- 
tions. 


Creatinine  and  Creatine.- 
HN  —  C  =  0 

HN  =  C 

I 

N  —  CH., 

CH3 

Creatinine 


0 

// 
H.N       C  —  OH 


HN==C 


N  — CH, 

I 
CH, 

Creatine. 


Creatinine  and  creatine  are  two  closely  related  compounds 


168  •    PHYSIOLOGICAL    CHEMISTRY 

occurring  in  the  urine.  Creatinine  is  the  anhydride  of  creatine. 
It  occurs  in  the  urine  of  adults  in  amounts  ranging  from  1-2 
grams  a  day.  On  a  diet  which  contains  no  creatinine,  the  amount 
is  quite  independent  of  the  amount  of  protein  in  the  food.  It  is 
thus  evident  that  it  is  not  an  end  product  of  the  metabolism  of 
food  proteins.  If  creatinine  is  present  in  the  food,  however,  (it 
is  found  in  meats,  meat  extracts,  etc.),  almost  all  of  the  amount 
ingested  reappears  in  the  urine,  and  thus  evidently  is  not  de- 
stroyed in  the  body.  On  a  creatinine  free  diet,  the  amount  of 
creatinine  in  the  urine  is  remarkably  constant  for  each  individ- 
ual,— about  7-11  mg.  of  creatinine  nitrogen  per  kilo  body  weight 
per  day  being  excreted.  This  value  is  called  the  creatinine  coef- 
ficient. 

Creatine  is  found  in  the  urine  of  children,  of  women  during 
pregnancy,  menstruation  and  after  childbirth,  and  in  the  urine 
of  adult  men  and  women  during  fasting.  At  this  time  the 
amount  of  creatinine  decreases,  the  total  of  creatine  -|-  creat- 
inine remaining  about  constant. 

If  creatine  is  taken  by  mouth,  little  or  no  increase  in  urinary 
creatinine  or  creatine  occurs,  a  very  singular  fact.  Creatine  oc- 
curs as  a  constituent  of  most  organs,  and  of  voluntary  muscle. 
There  are  about  120  grams  of  creatine  in  the  body  of  an  average 
adult.     Creatinine  also  may  be  obtained  from  muscles. 

The  fact  that  the  creatinine  excretion  is  independent  of  the 
protein  and  creatine  intake,  that  it  varies  with  age  and  sex,  and 
shows  a  fairly  constant  value  in  each  individual  has  led  to  the 
conclusion  of  Folin  that  creatinine  is  a  product  and  index  of  the 
endogenous  metabolism  of  the  tissues.  The  source  of  the  urinary 
creatinine  is  probably  the  creatine  of  the  tissues.  The  forma- 
tion of  creatinine  appears  to  be  independent  of  muscular  work, 
as  no  increase  is  observed  after  exercise.  Just  what  governs  the 
formation  of  creatinine  is  still  uncertain.  The  excretion  of 
creatinine  is  greater  during  the  day  than  at  night,  so  that  it  has 
been  suggested  its  formation  may  depend  in  some  way  on  the 
maintenance  of  muscle  tone,  but  this  point  is  as  yet  undecided. 

The  whole  subject  of  the  role  and  formation  of  the  creatine  of 


URINE  169 

the  tissues  and  the  formation  of  creatinine  is  still  in  a  very  un- 
satisfactory state,  although  it  appears  evident  that  these  sub- 
stances are  intimately  connected  with  endogenous  tissue  metab- 
olism itself, — with  the  actual  wear  and  tear  of  the  tissue  sub- 
stance. 

Inorganic  Constituents 

The  urine  contains  a  variety  of  inorganic  constituents 
such  as  chlorides,  sulphates,  phosphates,  carbonates,  etc., 
of  sodium,  potassium,  calcium  and  magnesium.  In  addition, 
various  others  occur.  There  are  traces  of  nitrates,  believed  to 
come  mainly  from  nitrates  in  the  drinking  water.  Iron  also  is 
found  in  traces,  about  8-10  mg.  per  day.  It  probably  is  both  in 
inorganic  and  organic  compounds.  Fluorides,  silicic  acid  and 
other  substances  also  occur,  as  well  as  accidental  constituents 
taken  with  the  food. 

Chlorides. — Of  the  inorganic  constituents,  chlorides  make  up 
the  largest  part.  Sodium  chloride  is  present  in  greatest  amount, 
and  the  total  chlorides  of  the  urine  usually  are  reported  as 
"sodium  chloride."  The  total  amount  averages  10-15  grams  of 
sodium  chloride  a  day,  but  it  may  vary  greatly,  the  variation  de- 
pending chiefly  on  the  amount  of  "salt"  in  the  diet.  Drinking 
much  water  will  increase  the  chloride  output.  The  excretion  is 
greater  during  activity  than  at  night.  The  amount  decreases  in 
some  diseases,  as  during  the  formation  of  exudates  in  pneumonia. 
When  reabsorption  takes  place  after  the  crisis,  the  retained 
chloride  reappears  in  the  urine.  It  is  interesting  that  sodium 
chloride  is  the  only  salt  present  in  an  ordinary  mixed  diet  in 
amounts  insufficient  for  the  body's  needs.  It  must  be  added  to 
the  food.  The  well  known  salt  craving  of  herbivorous  animals 
is  an  example  of  this.  Carnivorous  animals  obtain  enough  salt 
from  the  bodies  of  the  animals  they  devour. 

Some  chlorine  may  be  present  in  the  urine  in  organic  com- 
bination, but  if  so,  only  in  traces.  No  chlorine-containing  or- 
ganic constituent  of  the  body  is  known.  The  quantitative  esti- 
mation of  chlorides  is  given  in  the  laboratory  directions. 


170  PHYSIOLOGICAL    CHEMISTRY 

Phosphates. — The  urine  contams  about  0.5-1.2  grams  phos- 
phorous per  day,  but  none  of  this  is  in  the  form  of  free  phos- 
phorus. It  all  is  in  oxidized  form  as  phosphates,  a  portion  of 
which  are  present  as  mono-  or  di-sodium,  potassium,  calcium  or 
magnesium  phosphates,  some  probably  as  free  phosphoric  acid, 
and  some  in  ester-like  compounds  such  as  glycero-phosphoric 
acid,  etc.  The  amount  excreted  is  increased  by  a  protein  diet, 
since  the  phosphates  of  the  nucleoproteins,  lecithin,  the  phospho- 
proteins,  etc.,  are  the  main  source  of  urinary  phosphates.  Some 
inorganic  phosphates  also  occur  in  the  food  as  such.  The 
amount  in  the  urine  also  is  influenced  by  the  fact  that  phos- 
phates are  excreted  in  the  feces.  This  is  due  partly  to  failure  to 
absorb  them,  but  also  to  the  fact  that  phosphates  are  excreted 
into  the  intestine  and  eliminated  in  the  feces  chiefly  as  calcium 
phosphate.  On  an  average,  about  50-65%  of  the  total  phos- 
phorus is  excreted  in  the  urine,  the  remainder  in  the  feces.  Con- 
stipation causes  increase  in  urine  phosphorus,  and  decrease  in 
that  in  the  feces.  Diarrhea  has  the  opposite  effect.  The  phos- 
phates of  the  urine  are  sometimes  differentiated  into  alkali  phos- 
phates, alkaline  earth  phosphates,  and  organically  combined 
phosphates. 

In  pathological  conditions  the  phosphorus  excretion  is  in- 
creased when  there  is  increased  destruction  of  nuclear  material 
as  in  leucemia.  Also  in  starvation  the  bone  tissue  is  gradually 
drawn  upon  for  fuel,  and  increased  phosphate  elimination  re- 
sults. The  parathyroid  glands  also  appear  to  be  connected  in 
some  way,  as  yet  not  understood,  with  phosphate  excretion. 

Sulphates.— Sulphur  occurs  in  the  urine  in  various  forms. 
Three  classes  usually  are  recognized,  inorganic  sulphates, 
ethereal  sulphates  and  unoxidized  or  "neutral"  sulphur.  The 
total  amount  of  sulphur  excreted  per  day  is  variable,  but  usually 
is  somewhat  less  than  a  gram.  The  chief  source  of  urinary  sul- 
phate is  the  sulhpur  of  the  protein  molecule.  Most  of  this  is 
oxidized  in  the  body,  so  that  75-80%  of  the  total  sulphur  is  pres- 
ent as  inorganic  sulphate.  The  remainder  is  made  up  of  organi- 
cally bound  sulphate,  and  of  a  mixture  of  traces  of  several  com- 


URINE  171 

pounds  containing  sulphur  in  unoxidized  form,  such  as  sul- 
phocyanate  (which  we  already  have  met  as  a  constituent  of 
saliva),  cystin,  sulphides,  etc. 

Since  the  proteins  are  the  chief  source  of  urine  sulphur,  there 
is  a  general  parallelism  between  the  nitrogen  and  sulphur  excre- 
tion, which  often  runs  about  as  follows:  N:H2S04  =  5:1.  But 
this  is  by  no  means  a  constant  ratio. 

The  ethereal  sulphates  are  compounds  in  which  sulphuric  acid 
is  conjugated  with  various  phenols  produced  from  the  proteins 
by  putrefaction  in  the  intestine.  From  tryptophane,  indol  and 
skatol  are  produced  and  oxidized  in  the  body  to  indoxyl  and 
skatoxyl  which  are  then  combined  with  sulphuric  acid  to  make 
ethereal  sulphates.  The  synthesis  of  these  compounds,  as  in  the 
case  of  the  conjugated  glucuromates,  is  a  protective  measure, 
the  toxic  phenols  being  converted  into  relatively  non-toxic 
ethereal  sulphates. 

Variations  in  the  amount  of  protein  in  the  diet  cause  rela- 
tive as  well  as  absolute  variations  in  the  different  sulphur  constit- 
uents. If  the  amount  of  protein  in  the  food  is  decerased,  the  in- 
organic sulphates  fall  both  in  amount  and  in  per  cent  of  the  total 
sulphur  which  they  represent.  The  amount  of  neutral  sulphur 
remains  fairly  constant,  so  that  the  per  cent  rises.  Thus  neutral 
sulphur,  like  creatinine,  probably  is  largely  a  product  of  endog- 
enous tissue  metabolism,  since  it  is  independent  of  the  pro- 
tein intake  in  the  food.  Neutral  sulphur  is  increased  in  cystin- 
uria,  by  chloroform  and  after  taking  cyanides,  which  are  con- 
verted into  sulphocyanate.  Putrefaction  in  the  intestine  de- 
creases in  amount  on  a  low  protein  diet,  so  that  ethereal  sulphates 
decrease,  but  usually  not  so  rapidly  as  the  total  sulphur,  so  the 
percentage  increases  somewhat. 

Caxbonates  are  found  often  to  considerable  extent  in  the 
urine.  An  alkaline  reaction  of  the  urine  may  be  due  largely  to 
carbonates.  They  often  precipitate  in  alkaline  urine  and  are 
thus  found  in  the  sediment. 

Sodium,  potassium,  calcium  and  magnesium  are  present 
chiefly  as  chlorides,  sulphates,  phosphates  and  carbonates.    The 


172  PHYSIOLOGICAL    CHEMISTRY 

amounts  depend  much  on  the  quantity  of  each  present  in  the 
food.  In  the  ease  of  calcium  and  magnesium  much  may  be  lost 
in  the  feces.  About  1  gram  a  day  of  the  phosphates  of  these  two 
metals  is  found  in  the  urine.  Two  to  4  or  5  grams  each  of 
sodium  and  potassium  may  be  excreted  daily. 

Pathological  Constituents  of  the  Urine 

The  most  frequent  pathological  constituents  of  the  urine  are 
proteins,  carbohydrates,  acetone  bodies,  casts,  bacteria,  etc.  The 
study  of  pathological  urine  lies  properly  in  the  field  of 
pathology.  A  short  review  of  the  kinds  of  the  above  substances 
occurring  most  frequently  in  pathological  urine  has  been  in- 
cluded in  the  laboratory  chapter  on  the  urine,  together  with  the 
most  important  or  convenient  methods  for  detecting  and  estimat- 
ing them.  Certain  anomalies  of  metabolism  leading  to  the  ap- 
pearance of  these  substances  in  urine  will  be  discussed  in  the 
chapter  on  metabolism. 


CHAPTER  XII 
METABOLISM 

General. — The  body  in  some  respects  resembles  an  engine, — 
it  requires  fuel  to  carry  out  its  various  activities.  This  fuel  is 
"burned"  or  oxidized,  and  heat  or  mechanical  work  result.  In 
the  process,  waste  products  are  formed.  In  addition,  the  in- 
dividual parts  of  the  structure  are  continually  breaking  down 
and  being  repaired.  All  this  we  know  from  a  comparison  of  the 
' '  fuel ' '  or  food  substances  ingested,  and  the  waste  products  elim- 
inated. All  evidence  points  to  the  fact  that  extensive  chemical 
activity  is  going  on  in  the  tissues.  Materials  are  torn  t(3  pieces, 
chemically  speaking,  other  substances  are  built  up.  The  field 
covering  the  processes  going  on  in  the  tissues  is  known  as  metab- 
olism. This  does  not  include  digestion,  for  strictly  speaking, 
the  contents  of  the  alimentary  tract  are  not  in  the  body,  at  least 
not  in  the  tissues,  but  only  in  a  passageway  or  tube  running 
through  the  body.  The  study  of  metabolism  covers  the  history 
of  the  foodstuffs  from  the  time  of  their  absorption  to  the  point 
where  they,  or  the  products  formed  from  them  are  excreted  from 
the  body.  We  may  differentiate  various  fields  of  metabolism, 
such  as  that  of  proteins,  carbohydrates,  fats  or  inorganic  ma- 
terials, and  also  some  other  fields,  such  as  energy  exchange.  In 
addition,  we  may  study  the  general  metabolism  of  a  localized 
area,  such  as  metabolism  of  the  muscles,  liver,  etc. 

It  is  obviously  impossible  to  enter  the  tissues  and  observe  what 
is  going  on  in  them.  Our  observations  must  be  made  in  indirect 
ways,  and  conclusions  drawn  as  well  as  the  case  permits.  Vari- 
ous methods  for  the  study  of  metabolism  are  employed.  For  ex- 
ample, from  variations  in  the  constituents  of  the  urine  and  the 
amounts  of  the  different  substances  present,  from  observations 
made  on  excised  surviving  organs  through  which  blood  or  some 

173 


174  PHYSIOLOGICAL    CHEMISTRY 

similar  fluid  is  kept  circulating,  from  a  study  of  the  relationship 
between  oxygen  consumed  and  carbon  dioxide  eliminated 
(respiratory  quotient),  and  from  measurements  of  the  heat  pro- 
duced by  the  body  much  valuable  information  has  been  obtained 
about  the  processes  going  on  in  the  tissues. 

Protein  Metabolism. — The  metabolism  of  proteins,  and 
problems  connected  with  this  field  make  up  one  of  the  most  im- 
portant phases  of  metabolism.  The  reason  for  this  is  not  difficult 
to  divine.  Proteins  make  up  the  bulk  of  the  solid  material  of 
living  tissue.  Without  an  adequate  supply  of  them  in  the  diet, 
an  animal  will  die,  even  though  it  be  given  all  it  will  eat  of  the 
other  food  substances  such  at  fats,  carbohydrates,  salts,  etc. 
Evidently  proteins  have  some  role  to  play  which  cannot  be  filled 
by  the  other  classes  of  foods.  Since  the  other  foods  are  good 
fuels,  and  are  easily  burned  in  the  body,  the  importance  of  the 
proteins  seems  to  be  connected  with  furnishing  building  or  repair 
material  to  the  tissues,  or  supplying  certain  chemical  group- 
ings required  for  the  manufacture*  of  some  vitally  important 
products  manufactured  in  the  body  itself.  It  will  be  of  in- 
terest to  follow  the  proteins  and  the  products  formed  from  them 
in  their  passage  through  the  organism. 

In  the  digestive  tract  proteins  are  broken  down  by  enzymes 
into  amino  acids  and  in  this  form  they  are  absorbed  and  pass 
into  the  capillaries  of  the  villi,  and  thence  into  the  general  cir- 
culation by  way  of  the  portal  vein.  Until  a  few  years  ago  it  was 
a  matter  of  lively  dispute  whether  the  digestive  products  of  the 
proteins  entered  the  blood  in  the  form  of  amino  acids,  of  pro- 
teoses and  peptones,  or  were  rebuilt  in  the  intestinal  wall  to 
form  proteins.  It  had  never  been  possible  to  find  amino  acids 
or  proteoses  and  peptones  in  the  blood,  so  that  many  investigators 
believed  these  products  were  rebuilt  into  protein  before  passing 
into  the  blood.  One  by  one,  pieces  of  evidence  have  accumulated 
to  settle  this  question,  and  we  now  know  that  normally  the 
greater  part  at  least  of  the  amino  acids  produced  in  digestion 
gets  into  the  blood  as  such,  and  is  transported  to  the  tissues.  It 
has  been  shown  that  if  proteoses  and  peptones  are  injected  into 


METABOLISM  175 

the  blood,  enzymes  appear  in  the  blood  stream  capable  of  break- 
ing them  down.  No  such  enzymes  occur  in  the  blood  normally. 
Obviously  then,  proteoses  and  peptones  normally  are  not  present 
in  the  blood.  The  chief  difficulty  in  showing  that  free  amino 
acids  are  present  in  the  blood  lay  in  the  complex  character  of 
that  liquid,  and  its  high  content  of  protein.  During  the  diges- 
tion of  a  protein  meal  the  amount  of  amino  acids  absorbed  at 
any  one  time  would  be  very  small.  Also  the  flow  of  the  blood  is 
rapid,  so  that  any  increase  in  the  total  nitrogen  of  the  blood  would 
be  slight  and  thus  difficult  to  detect  in  the  presence  of  so  much 
other  nitrogenous  material.  Folin  and  his  co-workers  showed  an 
increase  in  the  non-protein  nitrogen  of  the  blood  after  a  pro- 
tein meal.  Abderhalden  demonstrated  that  amino  acids  are 
present  in  the  blood,  by  using  very  large  volumes  of  blood.  Van 
Slyke,  by  his  now  well  known  method  for  determining  small 
amounts  of  oc  amino  groups,  show^ed  a  marked  increase  after 
a  protein  meal,  and  Abel  and  his  co-workers  by  passing  the  cir- 
culating blood  of  a  living  animal  through  a  diffusion  device  suc- 
ceeded in  isolating  considerable  quantities  of  amino  acids  from 
the  blood.  The  problem  was  thus  solved.  The  proteins  of  the 
food  are  reduced  to  amino  acids  in  the  digestive  tract,  are 
absorbed  as  such,  and  transported  to  the  tissues.  It  has  been  re- 
ported that  a  portion  of  the  amino  acids  lose  their  amino  groups 
in  the  intestinal  wall.  Possibly  this  is  the  case.  This  statement 
also  has  been  contradicted,  however. 

The  further  fate  of  the  amino  acids  still  is  very  obscure. 
They  are  taken  up  by  the  tissues  from  the  blood.  Probably  cer- 
tain of  the  amino  acids  are  used  for  the  repair  of  cell  proteins, 
or  in  the  manufacture  of  cell  products.  There  appears  to  be 
little  or  no  storage  of  protein  material,  however,  for  the  nitrogen 
from  ingested  protein  reappears  in  the  urine  within  twenty-four 
hours  or  so  after  taking.  The  protein  structures  of  the  body  ap- 
pear to  increase  only  in  growth,  in  recovery  after  a  wasting  dis- 
ease, or  in  connection  with  increased  activity,  and  not  as  the  re- 
sult of  taking  an  excessive  supply  of  this  material. 

Various  modes  of  decomposition  are  possible  in  the  destruc- 


176  PHYSIOLOGICAL    CHEMISTRY 

tion  of  amino  acids.  The  amino  group  might  be  removed,  leaving 
a  fatty  acid,  or  oxidation  might  result  in  the  formation  of  an 
oxyacid  or  a  keto  acid ;  thus  from  alanine,  propionic  add,  lactic 
acid,  or  pja'uvic  acid  would  result. 

CH3  CH3  CH3  CH3 

I  I  i  I 

CHNH.  CH2  CHOH  C  =  0 

I  "  I  I  I 

COOH  COOH  COOH  COOH 

Alanine        Propionic  Acid    Lactic  Acid       Pyruvic  Acid 

Enzymes  are  known  which  can  produce  any  one  of  these  types  of 
reaction.  These  products  might  then  be  further  oxidized  to  COg 
and  HgO,  or  they  or  their  derivatives  could  be  utilized  in  the 
construction  of  other  substances  needed  by  the  tissues.  Pyruvic 
acid  and  the  corresponding  aldehyde  undoubtedly  play  an  im- 
portant part  in  the  destruction  of  more  than  one  amino  acid, 
and  the  resynthesis  of  other  substances.  The  property  of  de- 
aminizing  amino  acids  undoubtedly  is  possessed  by  all  cells.  The 
liver  plays  an  important  part  in  this  process  but  is  by  no  means 
the  only  place  where  it  occurs,  for  probably  all  living  tissues 
contribute. 

Amount  of  Protein  Required. — Nitrog-en  Balance.^ — Since 
protein  is  indispensable,  it  is  of  interest  to  know  how  much 
protein  is  needed  by  the  body.  Protein  is  an  expensive  article 
of  diet.  It  would  be  of  economic  advantage  to  eat  no  more  pro- 
tein than  necessary  for  the  maximum  efficiency  of  the  body. 
Excess  protein  is  not  stored  for  future  use,  but  is  broken  down, 
and  its  nitrogen  excreted  mainly  as  urea  within  a  comparatively 
short  period.  Ordinarily  aii  adult  is  in  a  condition  known  as 
nitrogen  equilibrium.  That  is,  he  excretes  just  the  amount  of 
nitrogen  that  he  takes  in  his  food.  His  body  is  neither  storing 
up  nor  depleting  its  store  of  nitrogenous  material  or  protein. 
Such  a  condition  of  exact  balance  does  not  always  exist,  how- 
ever. In  a  growing  child  or  in  an  adult  during  recovery  from 
a  wasting  disease,  or  even  during  periods  of  ''training"  or  un- 
wonted exercise,  the  body  may  lay  on  protein  tissue,^ — muscle  for 


METABOLISM  177 

example.  In  this  case  the  individual  will  be  excreting  less  nitro- 
gen than  he  receives  in  his  foods.  He  is  said  to  be  in  positive 
nitrogen  balance  since  he  is  laying  up  nitrogen  compounds.  In 
wasting  disease,  in  starvation,  or  on  insufficient  protein  diet  the 
loss  of  nitrogen  may  be  greater  than  the  intake  in  the  food.  This 
condition  is  known  as  negative  nitrogen  balance. 

It  is  a  curious  fact  that  nitrogen  balance  may  be  maintained 
on  widely  varying  levels  without  any  apparent  inconvenience. 
The  problem  of  the  necessary  amount  of  protein  in  the  diet  thus 
resolves  itself  into  the  problem, — on  what  amount  of  protein  can 
an  individual  be  kept  in  nitrogen  equilibrium;  a  further  phase 
of  the  question  will  be  to  determine  whether  minimum  protein 
is  desirable  from  a  physiologic  and  general  point  of  view. 

If  a  man  is  consuming  a  diet  containing  10  grams  of  protein 
nitrogen,  and  is  on  nitrogen  equilibrium  at  that  level,  suppose 
his  protein  ration  to  be  doubled,  so  that  he  will  be  taking  20 
grams  of  protein  nitrogen,  and  that  he  continue  on  this  diet. 
The  first  day  he  will  excrete  considerably  more  than  10  grams 
of  nitrogen,  but  not  the  entire  20  grams.  The  second  day  he  will 
excrete  still  more,  and  on  the  third  or  fourth  day  he  again  will 
be  in  nitrogen  equilibrium,  but  now  on  20  grams  nitrogen.  If 
this  is  now  cut  to  10  grams,  the  process  is  reversed,  and  in  three 
or  four  days  he  will  be  in  equilibrium  again  at  the  lower  level. 

An  ardent  advocate  of  a  low  protein  diet  is  Mr.  Horace 
Fletcher,  an  American  who  long  suffered  from  ill  health.  He 
greatly  reduced  both  the  total  quantity  of  food  taken,  and  the 
amount  of  protein  which  it  contained.  The  results  were  so  satis- 
factory that  a  study  of  the  problem  was  undertaken  by  various 
investigators.  Chittenden  carried  out  a  long  series  of  metabolism 
experiments  on  several  men.  Folin's  report  of  the  case  of  Dr. 
van  Sommeren  indicated  the  effects  on  the  quantitative  composi- 
tion of  the  urine  of  a  low  protein  diet.  (Folin's  "30  Normal 
Urines,"  an  analysis  of  30  24-hour  specimens  was  the  first  com- 
plete study  of  the  24-hour  output  of  normal  urine  constituents. 
This  furnishes  a  standard  for  comparison.)  These  results  al- 
ready have  been  referred  to.     Folin  himself  lived  on  a  starch 


178  PHYSIOLOGICAL   CHEMISTRY 

and  cream  diet  containing  only  about  6  grams  of  nitrogen  a 
day  for  several  days.  Chittenden  drew  the  conclusion  that 
the  most  desirable  amount  of  protein  for  an  average  sized 
adult  is  40  grams  per  day  or  somewhat  over  6  grams  of 
nitrogen.  This  is  much  less  than  the  standards  previously 
recommended.  Voit,  for  example,  basing  his  conclusions  on  the 
amount  of  protein  consumed  per  capita  in  several  European 
cities,  recommended  118  grams  of  protein  (19  grams  nitrogen) 
as  the  proper  amount,  and  most  other  standards  were  of  this  gen- 
eral magnitude. 

It  has  been  shown  that  nitrogen  equilibrium  may  be  estab- 
lished on  a  level  even  lower  than  that  of  Chittenden.  Thus 
Thomas  reduced  his  nitrogen  to  2.2  grams  a  day  (about  15-20 
grams  protein) .  In  fact  on  a  low  protein  intake  the  protein  evi- 
dently is  used  more  economically  by  the  body.  But  the  problem 
remains, — is  so  low  a  level  of  protein  intake  desirable  or  safe? 
Some  evidence  has  accumulated  on  this  point.  Certain  tribes  in 
India  who  live  on  low  protein  diet  have  been  observed  to  be  less 
efficient  and  to  possess  less  endurance  than  neighboring  tribes 
of  meat  eaters.  Races  living  in  cold  climates  usually  live  on 
a  high  protein  diet,  and  such  races  display  great  endurance. 
The  whole  experience  of  the  human  race  seems  to  have  tended 
toward  a  fairly  high  protein  diet.  If  low  protein  intake  resulted 
in  greater  efficiency,  it  is  only  reasonable  to  suppose  that  races 
or  nations  living  on  low  protein  rations  would  have  dominated 
over  others.  From  the  experiments  of  Chittenden  and  others  it 
appears  possible  to  maintain  a  group  of  individuals  for  several 
months  on  a  low  protein  diet,  apparently  with  good  results  and 
increased  efficiency  and  health.  It  might  be  suggested,  however, 
that  such  favorable  results  may  be  due  in  part  to  the  carefully 
regulated  living,  wisely  directed  exercise  and  wholesome  food 
of  the  subjects,  for  it  is  an  unfortunate  but  incontrovertable 
fact  that  many  of  us  are  sadly  lax  in  the  ordering  of  such  im- 
portant factors  in  our  existence  as  exercise,  proper  food,  and 
general  healthful  living.  From  experiments  of  but  a  few 
months'  duration  it  is  dangerous  to  draw  conclusions  of  so  im- 


METABOLISM  179 

portant  and  far-reaching  a  nature.  It  is  possible  that  on  mini- 
mum protein  intake,  reserve  stores  of  important  but  seldom 
needed  materials  might  be  seriously  depleted,  thus  reducing  the 
body's  power  of  resisting  disease  or  meeting  emergency  demands. 
In  this  connection,  the  work  of  Haecker  is  most  interesting.  A 
herd  of  cattle  was  kept  for  about  three  years  on  low  protein 
diet.  The  first  two  years  all  went  well.  In  the  third  year  the 
animals  showed  lessened  resistance  to  the  inroads  of  disease,  and 
finally  became  so  ill  that  the  experiment  was  discontinued. 

In  summary,  perhaps  the  older  standards  of  about  120 
grams  protein  are  unnecessarily  high.  A  compromise  on  per- 
haps 90  grams  of  protein  for  the  average  adult  has  been  sug- 
gested as  the  most  satisfactory  solution  of  the  problem. 

The  fact  that  nitrogen  equilibrium  can  be  maintained  on  so 
loAv  a  level  as  2-3  grams  N  a  day  makes  it  seem  possible  that  the 
breaking  down  of  the  protein  tissues  takes  place  to  a  much 
smaller  extent  than  was  supposed.  Another  and  a  more  prob- 
able explanation  of  this  fact  is  that  much  of  the  material  pro- 
duced in  the  destruction  of  protein  tissues  is  used  again  in  the 
body  to  rebuild  the  destroyed  tissues. 

The  7naximum  amount  of  protein  on  which  nitrogen  balance 
may  be  maintained  is  of  little  practical  interest.  It  will  be 
limited  by  the  ability  of  the  organism  to  absorb  protein  products 
from  the. digestive  tract.  Excess  protein  is  simply  destroyed  in 
the  organism.  In  addition  to  the  economic  disadvantage  of  so 
expensive  a  diet,  unnecessary  strain  is  put  on  the  excretory  or- 
gans in  disposing  of  the  excess  of  urea  and  other  end  products 
formed.  It  is  an  interesting  fact  that  increased  muscular  ac- 
tivity does  not  increase  nitrogen  excretion  if  the  animal  is  well 
nourished.  The  animal  burns  carbohydrates  and  fats,  and  de- 
stroys no  more  protein. 

Thijs  far  we  have  spoken  only  of  protein  need.  In  so  doing, 
we  really  are  considering  amino  acid  need,  for  the  proteins  are 
broken  down  to  amino  acids  in  digestion.  Since  proteins  contain 
widely  varying  proportions  of  the  different  amino  acids,  and  a 
given  protein  often  lacks  one  or  more  of  these  compounds,  it  is 


180  PHYSIOLOGICAL    CHEMISTRY 

not  surprising  that  different  proteins  vary  greatly  in  their 
usefulness  and  adequacy  in  the  diet.  The  amount  of  protein 
required  thus  is  dependent  on  the  kind  of  protein.  If  the  body 
requires  a  definite  amount  of  a  particular  amino  acid,  that  amino 
acid  must  be  present  in  the  food  protein  unless  the  body  is 
capable  of  constructing  it  in  its  own  workshop  from  other  ma- 
terial such  as  ammonia  and  residues  of  other  amino  acids,  of 
carbohydrates  or  other  substances.  Most  interesting  results  have 
been  obtained  in  this  field. 

Gelatine  lacks  tyrosine  and  tryptophane.  Although  gelatine 
is  a  protein,  if  an  otherwise  adequate  diet  is  fed  containing  gela- 
tine as  the  sole  protein  constituent,  the  animal  will  die  as  surely 
as  if  he  were  receiving  no  protein.  Addition  of  these  missing 
amino  acids  to  the  diet,  either  directly  or  by  adding  a  protein 
which  contains  them  will  remedy  the  difficulty,  and  make  the 
diet  adequate.  Osborne  and  Mendel,  and  McCollum  have  con- 
tributed greatly  to  our  knowledge  in  this  field.  Zein,  a  protein 
from  corn,  contains  no  tryptophane  or  lysine.  On  an  otherwise 
adequate  diet  containing  zein  as  its  sole  protein,  a  young  animal 
declines  and  dies.  On  adding  tryptophane  to  such  a  diet,  the 
animal  no  longer  loses  weight.  It  maintains  about  the  same  body 
weight.  Apparently  tryptophane  is  necessary  for  body  main- 
tenance. But  on  such  a  diet  the  animal  does  not  grow.  If  lysine 
be  added  along  with  the  tryptophane,  the  animal  now  grows 
almost  at  a  normal  rate.  Lysine  thus  appears  to  be  necessary  for 
body  growth.  From  the  above  discussion  it  is  evident  that  the 
body  cannot  synthesize  tyrosine,  tryptophane,  or  lysine,  at  least 
in  amounts  sufficient  for  its  needs.  Some  of  the  a:mino  acids  ap- 
parently can  be  built  up  by  the  body.  Thus  after  feeding  ben- 
zoic acid,  glycocoll  will  be  excreted  in  hippuric  acid  in  quantities 
too  large  to  be  accounted  for  by  the  available  glycocoll  in  the 
organism.  Probably  some  other  amino  acids,  among  them  pro- 
line also  can  be  built  up  by  the  body. 

The  fact  that  maintenance  is  possible  on  a  zein-tryptophane 
protein  ration  (lacks  lysine)  but  not  growth,  which  would  entail 
formation  of  new  protein,  may  indicate  that  the  breaking  down 


METABOLISM 


181 


of  proteins  in  the  tissues  is  not  general,  but  for  the  purpose  of 
supplying  some  amino  acid  required  for  the  manufacture  of  a 
necessary  substance.  Otherwise,  it  would  be  difficult  to  under- 
stand why  the  body  can  build  enough  protein  to  repair  that 
broken  down,  but  no  more  for  growth  unless  lysine  also  is  fed. 

The  problem  of  protein  requirement  thus  is  resolved  into  a 
problem  of  amino  acid  requirement,  limited  on  the  one  hand  by 
the  body's  needs,  and  on  the  other  hand  by  its  ability  to  con- 
struct amino  acids  itself. 

Since  amino  acids  are  the  real  requirement  of  the  body,  one 
would  expect  it  to  be  possible  to  supply  an  animal's  protein  re- 
quirement solely  by  a  mixture  of  amino  acids.  Such  experiments 
have  been  attempted  and  their  results  point  in  general  to  this 
conclusion.  It  may  be  noted,  however,  that  the  difficulty  of 
experiments  of  this  sort  is  much  increased  by  the  unpalatable 
nature  of  such  a  mixture,  so  that  positive  results  have  not  been 
always  the  rule.  Dogs  fed  on  such  mixtures  often  refuse  to  eat, 
and  if  fed  by  a  stomach  tube,  frequently  vomit.  In  a  German 
laboratory,  an  attempt  by  one  of  the  assistants  to  live  for  a 
period  upon  a  mixture  of  amino  acids  and  the  building  stones 
of  the  fats  and  carbohydrates  as  well,  was  abandoned  after  the 
first  attempted  meal  as  a  result  of  the  unappetizing  nature  of 
the  mixture.  By  injection  of  protein  decomposition  products 
into  the  blood,  it  has  been  possible  to  maintain  an  animal  on 
nitrogen  equilibrium. 

The  role  played  by  the  individual  amino  acids  in  the  organism 
is  still  obscure.  Probably  certain  of  them  are  used  as  repair 
material  for  the  tissue  proteins.  Others  may  be  employed  for  the 
manufacture  of  important  products  of  internal  secretion,  whereas 
excess  of  these,  and  such  acids  as  are  not  required,  undoubtedly 
are  deaminized  and  the  residues  either  used  for  constructing 
other  compounds,  or  burned  as  fuel,  as  are  the  fats  and  car- 
bohydrates. 

The  composition  of  the  proteins  of  the  tissues  is  remarkably 
constant,  and  quite  independent  of  the  nature  of  the  food  pro- 
tein.    A  horse  was  bled  to  remove  much  blood  protein.     This 


182  PHYSIOLOGICAL   CHEMISTRY 

the  body  was  obliged  to  replace.  The  horse  was  fed  at  the  time 
on  gliadin,  a  grain  protein  containing  a  large  percentage  of 
glutamic  acid.  The  blood  proteins  contain  less  than  a  quarter 
as  much  glutamic  acid  as  does  gliadin,  but  they  were  regenerated, 
and  showed  no  variation  from  their  normal  composition. 

The  proteins  of  each  individual  tissue,  and  of  each  different 
animal  undoubtedly  are  specific,  that  is  those  from  different 
sources  differ  slightly  in  composition.  It  has  been  suggested  that 
possibly  they  are  the  substances  which  transmit  species  charac- 
teristics, since  they  vary  in  different  animals,  whereas  most  of 
the  other  body  compounds,  such  as  salts,  carbohydrates  enzymes, 
nucleic  acids,  etc.,  are  practically  identical  in  different  animals. 
This  is  in  the  realm  of  speculation,  however. 

Carbohydrate  Metabolism. — A  field  of  interest  second  only 
to  that  of  protein  metabolism  is  the  metabolism  of  carbohydrates. 
Carbohydrates  make  up  a  large  and  important  part  of  our  food, 
and  although  they  play  a  much  less  conspicuous  role  as  constitu- 
ents of  body  tissue,  they  are  excellent  fuels,  and  furnish  the  body 
with  a  considerable  proportion  of  the  material  burned  for  the 
maintenance  of  body  temperature  and  the  performance  of  me- 
chanical work. 

The  various  carbohydrates  of  the  food  such  as  starches,  dex- 
trins,  disaccharides,  etc.,  are  reduced  to  monosaccharides  by  the 
digestive  enzymes,  and  as  such  are  absorbed  and  pass  into  the 
blood  stream.  What  is  their  further  fate?  The  monosaccha- 
rides have  been  shown  to  be  present  in  the  blood  stream  as  such, 
and  not  combined  or  united  with  any  other  substance,  at  least 
in  more  than  the  most  unstable  union.  Dialysis  of  blood  against 
dextrose  solutions  of  various  strengths  has  shown  that  the  blood 
sugar  evidently  is  free. 

The  blood  from  the  intestine  is  gathered  into  the  portal  vein 
and  passes  to  the  liver  and  here  begins  the  story  of  its  utilization 
in  the  body.  Claude  Bernard,  a  French  scientist,  discovered  in 
the  liver  a  substance  to  which  he  gave  the  name  glycogen.  This 
substance  is  a  polysaccharide,  and  on  hydrolysis  yields  glucose. 
Glycogen  occurs  in  places  other  than  the  liver,  for  example,  the 


METABOLISM  183 

muscles  also  may  contain  it.  It  appears  that  glycogen  is  a 
reserve  supply  material  which  serves  to  store  up  sugar  for  the 
organism.  In  case  of  need,  the  glycogen  is  broken  down,  and  fur- 
nishes the  tissues  with  a  supply  of  glucose  for  fuel.  The  amount 
of  glycogen  which  the  liver  and  muscles  can  store  is  limited, 
however.  About  150  grams  is  the  maximum  amount  which  either 
of  these  tissues  can  lay  up.  Since  glycogen  is  a  reserve  fuel  for 
the  body,  it  is  called  upon  in  case  of  need  and  conditions  re- 
quiring the  body  to  call  on  its  reserves  will  cause  a  diminution 
in  the  glycogen.  Liver  glycogen  appears  to  be  particularly 
available  for  immediate  use.  Hard  work,  starvation,  exposure 
to  cold  and  various  other  conditions  will  greatly  reduce  the 
amount  of  glycogen  in  the  liver,  and  also  in  the  muscles.  The 
sources  from  which  glycogen  may  be  built  up  will  be  discussed 
at  a  later  point  in  this  chapter. 

The  breaking  down  of  liver  glycogen  has  been  shown  to  be 
influenced  by  a  center  in  the  medulla.  Injury  to  this  center, 
which  may  be  brought  about  in  rabbits  by  forcing  a  steel  pencil 
into  the  brain  in  front  of  the  occipital  prominence  in  such  a 
way  that  the  floor  of  the  fourth  ventricle  is  pierced,  causes  sugar 
to  appear  in  the  urine.  The  percentage  of  sugar  in  the  blood 
rises  much  above  normal.  If  the  animal  is  killed  and  the  liver 
examined,  it  will  be  found  to  contain  only  a  trace  of  glycogen. 
Evidently  impulses  from  this  region  of  the  medulla  cause  the 
conversion  of  liver  glycogen  into  glucose.  Overstimulation  of 
this  center  results  in  flooding  the  blood  with  sugar.  The  kidneys 
are  so  regulated  that  they  keep  a  constant  percentage  of  sugar 
in  the  blood.  Any  excess  over  this  amount  is  excreted  in  the 
urine.  It  is  evident  that  puncture  of  the  medulla  does  not 
destroy  the  sugar  center,  but  only  irritates  it,  for  the  glycosuria  is 
temporary,  and  passes  off  after  a  short  time.  The  center  is  con- 
sidered to  be  a  true  reflex  center,  and  may  be  stimulated  by 
afferent  impulses.  Thus  if  the  vagus  is  severed  and  the  central 
end  stimulated,  sugar  appears  in  the  urine.  If  the  splanchnics 
are  cut,  there  is  no  glycosuria  after  puncture.  Thus  evidently, 
it  is  only  liver  glycogen  which  is  affected  by  puncture,  and  the 


184  PHYSIOLOGICAL.    CHEMISTRY 

impulses  are  carried  to  the  liver  by  the  splanchnics.  Glycosuria 
produced  by  ''diabetic  puncture"  is  known  as  "puncture  dia- 
betes." 

A  second  factor  Avhich  apparently  is  connected  with  the  con- 
trol of  liver  glycogen  is  the  secretion  of  the  suprarenals,  adrena- 
line. If  adrenaline  is  injected,  an  increase  of  blood  sugar 
occurs,  which  has  its  source  in  liver  glycogen,  since  no  glycosuria 
results  if  the  glycogen  supply  of  the  liver  has  been  exhausted. 
It  has  been  suggested  that  the  action  of  the  sugar  center  in  the 
medulla  is  by  way  of  the  suprarenals.  This  problem  is  still  in 
doubt ;  it  is  probable  that  the  sugar  center  works  by  direct  stimu- 
lation of  the  liver  cells,  but  is  aided  and  supplemented  by  the 
adrenaline  secreted  from  the  suprarenals. 

The  pancreas  has  been  shown  to  play  an  important  part  in  the 
utilization  of  sugar  by  the  body.  This  is  connected  with  the 
burning  of  the  sugar  as  fuel  and  also  with  the  storage  of  gly- 
cogen in  the  liver.  If  the  pancreas  of  an  animal  is  removed, 
sugar  appears  in  the  urine,  and  the  amount  of  blood  sugar  rises 
much  above  the  normal.  Very  little  liver  glycogen  is  stored  up. 
The  explanation  of  these  facts  occupied  a  great  many  years,  and 
many  points  are  still  obscure.  It  was  found  that  if  even  a  small 
portion  of  pancreas  tissue  is  grafted  under  the  skin  and  the 
blood  vessels  and  nerves  of  the  fragment  left  intact,  extirpation 
of  the  remainder  of  the  gland  does  not  cause  glycosuria.  The 
action  of  the  pancreas  evidently  is  independent  of  the  pancreatic 
juice  secreted  into  the  intestine.  If  this  transplanted  portion  of 
the  pancreas  subsequently  is  removed,  hyperglycemia  (excess 
sugar  in  the  blood)  and  glycosuria  appear.  Only  within  the 
last  few  years  has  fairly  conclusive  evidence  been  obtained  that 
the  pancreas  produces  a  substance  which  is  given  off  into  the 
blood, — an  "internal  secretion,"  without  which  the  tissues  are 
unable  to  use  glucose.  If  this  substance  is  lacking,  the  amount 
of  glucose  in  the  blood  increases,  and  the  excess  is  excreted  in 
the  urine.  It  often  has  been  affirmed  that  the  "Islands  of 
Langerhans,"  small  groups  of  certain  cells  present  in  the 
pancreas,  are  responsible  for  the  production  of  this  important  in- 


METABOLISM  185 

ternal  secretion.  The  evidence  for  this  is  not  conclusive,  how- 
ever, and  it  is  still  uncertain  where  in  the  gland  the  substance 
is  formed. 

Still  another  factor  is  concerned  in  the  control  of  glucose 
utilization  in  the  body,  and  that  is  the  influence  of  the  kid- 
neys. It  has  been  stated  that  the  kidneys  are  ' '  set "  to  retain  a 
definite  percentage  of  sugar  in  the  blood.  The  kidneys  may  be 
injured  by  the  injection  of  the  drug  phloridzin.  There  is  no 
increase  in  the  level  of  blood  sugar,  but  sugar  appears  in  the 
urine  for  several  hours.  The  amount  of  sugar  in  the  blood  falls 
below  the  normal.  The  mechanism  of  the  process  is  undecided. 
Possibly  the  excretion  of  glucose  is  an  active  process,  and  not 
simply  the  passive  action  of  a  dam  to  keep  back  a  certain  amount 
of  sugar.  In  this  case  phloridzin  might  act  by  stimulating  the 
excretion  of  sugar  by  the  kidneys.  It  is  of  interest  in  this  con- 
nection that  the  drug  causes  marked  degeneration  of  kidney 
epithelium.  There  is  some  reason  to  believe  that  phloridzin 
causes  excretion  of  glucose  in  organs  other  than  the  kidneys. 

Much  speculation  has  been  expended  on  the  cause  of  diabetes 
in  man  and  its  possible  relationships  to  one  or  other  of  the  forms 
of  experimental  diabetes  discussed,  i.e.,  puncture  diabetes,  pan- 
creatic diabetes  or  phloridzin  diabetes.  Obviously  it  is  not 
analogous  to  the  last  form,  for  the  disease  is  accompanied  by 
increased  sugar  content  of  the  blood.  Clinicians  generally  are  of 
the  opinion  that  it  is  closely  allied  to  pancreatic  diabetes.  Evi- 
dence, however,  is  not  absolutely  conclusive  as  yet,  though  it  is 
generally  believed  that  lesions  in  the  pancreas  are  usually  if  not 
always  the  cause  of  the  disorder. 

A  review  of  the  foregoing  discussion  will  emphasize  the  fact 
that  the  internal  factors  regulating  carbohydrate  metabolism  in 
the  body  include  a  center  in  the  medulla,  presiding  over  the 
conversion  of  liver  glycogen  into  blood  sugar,  the  internal  secre- 
tion of  the  suprarenals,  operative  in  a  similar  way,  the  internal 
secretion  of  the  pancreas,  without  which  sugar  cannot  be  burned 
by  the  cells,  and  the  nature  of  the  kidney,  which  regulates  the 
level  of  sugar  in  the  blood. 


186  PHYSIOLOGICAL   CHEMISTRY 

Various  forms  of  temporary  glycosuria  are  known.  Thus, 
after  excessive  exercise,  during  great  agitation  or  mental  strain 
such  as  often  is  experienced  by  students  taking  a  difficult  ex- 
amination (emotional  glycosuria),  or  after  taking  excessive 
amount  of  simple  carbohydrates  (alimentary  glycosuria)  sagar 
may  appear  in  the  urine.  In  the  case  of  the  first  two  conditions, 
the  glycosuria  is  believed  to  depend  on  a  production  of  adren- 
aline, which  is  known  to  be  secreted  at  times  of  great  exertion  or 
emotional  stress,  a  logical  process,  since  at  such  times  the  mus- 
cles are  apt  to  need  an  increased  supply  of  fuel  for  use  in  pos- 
sible pursuit,  flight,  or  combat. 

An  important  phase  of  carbohydrate  metabolism  is  concerned 
with  the  ultimate  fate  of  the  glucose  which  is  burned  as  fuel. 
How  and  where  does  the  burning  or  oxidizing  take  place,  and 
how  is  it  controlled?  There  is  still  much  uncertain  ground  in 
this  field,  though  much  progress  has  been  made.  Various 
methods  have  been  employed  to  throw  light  on  the  problem.  In- 
teresting results  have  developed  from  a  study  of  the  Respiratory 
Quotient.  This  term  is  used  for  the  ratio  between  the  amount  of 
carbon  dioxide  excreted  in  the  respired  air,  and  the  amount  of 

CO 
oxygen  consumed,  and  is  indicated  as  ^r— ^ .  If  glucose  is  oxidized 

to  CO2  and  water  a  certain  amount  of  oxygen  is  consumed. 
CeH,,0e+6  0,^6  C0,+6  H3O. 

For  every  molecule  of  CO2  produced,  one  molecule  of  O2  is  used 

up.    There  already  is  enough  0  in  the  carbohydrate  to  take  care 

of  the  hydrogen  present.     Thus  the  volume  of  O2  consumed 

CO 
equals  the  volume  of  CO2  produced,  and  7^— ^  1-     This  will 

be  true  in  the  body  as  well  as  elsewhere  provided  the  oxidation 
of  the  carbohydrate  to  CO2  and  HgO  is  complete.  If  a  fatty 
acid  is  burned  in  the  same  way,  the  respiratory  quotient  is  less 
than  one.    It  does  not  contain  sufficient  oxygen  to  take  care  of 


METABOLISM  187 

even  the  hydrogen.     Thus  part  of  the  oxygen  consumed  is  ex- 

CO 
creted  as  water,  and  in  the  expression  -p^,  the  denominator  is 

'-'2 
larger,  and  the  ratio  is  less  than  one  (about  0.7).  In  the  ease  of 
amino  acids  (from  the  proteins)  the  value  lies  between  those  for 
carbohydrates  and  fats.  By  actual  measurement  of  the  amount 
of  CO2  excreted  by  an  animal,  and  the  amount  of  O2  consumed 
it  is  possible  to  draw  conclusions  as  to  the  kind  of  material 
Avhich  the  body  is  burning. 

Further  evidence  of  the  fate  and  history  of  glucose  in  the 
body  has  been  obtained  by  a  study  of  the  various  experimental 
glycosurias  described  above. 

Although  much  labor  has  been  expended  to  clear  up  the 
exact  mechanism  of  the  burning  of  glucose  in  the  muscles,  very 
little  is  known  of  the  steps  in  the  process.  We  are  sure  that 
glucose  serves  as  fuel  for  the  muscles  from  evidence  of  various 
sorts  but  only  in  the  presence  of  a  substance  produced  in  the 
pancreas.  It  has  been  suggested  that  glyceric  aldehyde  COH — 
CHOH — CHaOH  is  an  intermediate  stage  in  the  process  of 
burning  sugar,  and  that  this  is  converted  next  into  alcohol  CH3 
CHgOH  and  CO2,  but  this  has  not  been  proven.  The  diabetic 
is  unable  to  use  glucose,  but  the  exact  reason  for  the  failure  of 
the  tissues  to  use  sugar  is  unknown.  It  is  probable  that  in  some 
cases  at  least,  the  necessary  internal  secretion  of  the  pancreas  is 
wanting,  but  this  only  brings  us  a  step  nearer  the  solution  with- 
out actually  furnishing  it.  The  diabetic  is  still  capable  of  per- 
forming oxidations,  for  many  substances  other  than  glucose  still 
are  oxidized  with  ease.  It  has  been  suggested  that  the  difficulty 
is  in  the  first  attack  of  cleavage  on  the  sugar  molecule,  but  there 
is  some  evidence  which  does  not  bear  out  this  idea.  We  shall 
have  to  await  the  final  solution  of  the  problem. 

Sources  of  Glycogen. — The  study  of  the  sources  from  w'hich 
glycogen  can  be  built  up  in  the  body  has  been  greatly  facilitated 
by  the  knowledge  of  the  experimental  glycosurias.  It  is  of  in- 
terest to  know  what  substances  are  glycogen  formers  in  the  body. 
One  method  of  study  is  to  render  an  animal  as  nearly  as  possible 


188  PHYSIOLOGICAL    CHEMISTRY 

glycogen  free,  and  then  to  feed  the  substance  to  be  investigated. 
The  animal  then  may  be  killed  and  the  glycogen  content  of  liver 
and  muscles  estimated.  If  glycogen  is  present  in  quantity  it 
either  will  have  been  formed  from  the  material  fed,  or  indirectly, 
if  this  food  has  ' '  spared ' '  or  been  burned  in  place  of  some  other 
glycogen  former.  A  second  method  is  to  render  the  animal  dia- 
betic by  puncture,  phloridzin  or  other  means.  A  substance  then 
may  be  fed,  and  the  amount  of  sugar  in  the  urine  estimated.  An 
increase  will  indicate  that  the  substance  fed  is  a  glycogen  or 
rather  glucose  former,  provided  the  possible  origin  of  the  excess 
glucose  from  body  constituents  is  excluded.  To  render  an  animal 
glycogen  free,  it  may  be  made  to  fast  for  some  time,  to  do  work, 
it  may  be  subjected  to  cold,  or  thrown  into  convulsions  by  giving 
strychnine.  A  combination  of  methods  usually  gives  more  satis- 
factory^ results  than  any  single  method.  A  method  of  study  de- 
pending upon  quite  different  technique  may  be  used.  The  liver 
may  be  excised,  and  kept  supplied  with  a  circulating  medium, 
either  blood  or  some  other  fluid.  The  substance  to  be  studied 
then  may  be  introduced  into  the  circulating  fluid,  and  the  gly- 
cogen content  of  the  liver  later  determined. 

It  has  been  found,  as  might  be  expected,  that  glucose  forms 
glycogen.  Any  substance  which  will  form  glucose  in  the  body, 
thus  also  will  be  a  possible  glycogen  former.  Fructose,  also, 
and  to  some  extent  galactose  form  glycogen.  On  hydrolysis,  this 
glycogen  is  converted  into  glucose  and  not  into  the  sugar  from 
which  it  was  produced.  This  is  an  interesting  fact,  as  it  is  an 
example  of  a  conversion  of  one  monosaccharide  into  another  in 
the  body.  Naturally  all  carbohydrates  which  are  digested  to 
monosaccharides  in  the  alimentary  tract  thus  will  be  sources  of 
glycogen.  It  has  been  shown,  however,  that  there  are  sources  of 
glycogen  other  than  the  carbohydrates. 

If  an  animal  is  made  diabetic  by  removal  of  the  pancreas  or 
in  some  other  way,  glucose  appears  in  the  urine.  It  continues  to 
be  excreted  when  the  glycogen  supplies  of  the  body  have  been 
exhausted.  If  protein  is  fed  to  such  an  animal,  the  amount  of 
glucose  in  the  urine  increases.     There  is  a  certain  parallelism 


METABOLISM  189 

between  the  amount  of  glucose  (dextrose)  excreted  and  the 
amount  of  nitrogen  in  the  urine.  This  is  known  as  the  D :  N 
ratio.  All  the  protein  is  not  converted  into  glucose.  Portions 
of  some  of  the  amino  acids  are  destroj'ed  or  converted  into  other 
substances,.  Sixty  grams  of  glucose  is  considered  the  maximum 
amount  which  the  body  can  produce  from  100  grams  of  protein 
(this  contains  ca.  16  grams  nitrogen).  A  ratio  of  D  :  N=60 :  16 
or  about  1 :3.7  would  indicate  that  none  of  the  glucose  produced 
from  protein  was  being  burned  by  the  body,  in  other  words  a 
complete  failure  on  the  part  of  the  tissues  to  burn  glucose.  3.7 
is  thus  known  as  the  fatal  ratio.  It  must  be  observed,  however, 
when  the  animal  is  on  a  carbohydrate  free  diet,  as  otherwise  the 
ratio  may,  of  course,  be  still  higher.  By  observing  the  D-N  ratio 
when  various  amino  acids  were  fed,  it  has  been  shown  beyond 
doubt  that  some  amino  acids  are  converted  completely  into  glu- 
cose in  the  body,  some  others  only  partially.  There  is  thus  no 
question  of  the  formation  of  glucose  (and  glycogen)  from  the 
carbon  chain  of  the  amino  acids,  and  thus  indirectly  from  the 
proteins. 

Very  little  glycogen  is  formed  from  fat.  The  glycerine  por- 
tion may  be  converted  into  glucose  but  the  fatty  acids  do  not 
appear  to  be  converted  into  glucose. 

Metabolism  of  Fats. — The  fats  are  digested  in  the  alimen- 
tary tract  and  absorbed  in  the  form  of  fatty  acids  and  soaps.  In 
this  process  the  bile  salts  play  an  important  role.  In  the  cells 
of  the  villi,  how^ever,  a  re-synthesis  of  neutral  fat  takes  place, 
and  at  least  most  of  the  fatty  acids  and  glycerine  are  recombined, 
and  poured  into  the  blood  stream  by  way  of  the  thoracic  duct. 
Fats  are  stored  away  in  a  variety  of  places, — subcutaneously,  in 
the  intramuscular  spaces,  around  the  abdominal  viscera  and 
elsewhere.  There  is  practically  no  limit  to  the  amount  which 
may  be  laid  away.  This  is  in  sharp  contrast  to  the  non-storage 
of  excess,  protein,  and  to  the  limited  glycogen  reserves.  Fat  is 
a  reserve  fuel,  and  it  also  protects  the  body  from  loss  of  heat, 
like  a  subcutaneous  blanket,  for  it  is  a  poor  conductor. 

If  an  animal  is  fed  excessive  amounts  of  carbohydrate  food, 


190  PHYSIOLOGICAL   CHEMISTRY 

it  will  lay  on  reserves  of  fat.  There  is  evidence  thus  that  carbo- 
hydrates may  be  converted  into  fat  in  the  body,  although  it 
seems  that  the  reverse  process  does  not  take  place,  at  least  it  is 
very  questionable.  Since  amino  acids  may  be  converted  into 
carbohydrates,  they  also  may  be  fat  formers. 

Light  has  been  thrown  upon  the  mechanism  of  fat  oxidation 
in  the  body  in  various  ways.  Ordinarily  the  fatty  acids  are 
burned  completely  to  CO,  and  0,.  By  introducing  into  the  or- 
ganism compounds  in  which  a  fatty  acid  side  chain  is  attached 
to  a  benzene  ring,  the  last  step  in  this  destruction  of  the  side 
chain  is  prevented  and  the  nature  of  the  resulting  substance  may 
be  studied.  Such  compounds  in  which  the  side  chain  has  an 
even  number  of  carbon  atoms  are  oxidized,  and  the  side  chain 
destoyed  with  the  exception  of  hvo  carbon  atoms.  From 
phenylbutyric  acid,  phenyl  acetic  is  produced.  This  substance 
is  conjugated  with  glycoeoll  and  excreted  as  phenyl  aceturic 
acid,  a  compound  of  phenyl  acetic  and  glycoeoll  analogous  to  hip- 
puric  acid  (q.v.).  If  the  original  side  chain  contains  an  uneven 
number  of  carbon  atoms,  the  side  chain  is  oxidized  away,  and 
benzoic  acid  remains,  which  is  conjugated  with  glycoeoll  and 
excreted  as  hippuric  acid.  From  these  facts  it  is  evident  that 
the  side  chains  are  oxidized  so  that  not  one  carbon  atom,  but 
two  are  removed  at  each  step. 

Thus  it  is  believed  that  the  fatty  acids  from  the  fats  also  are 
oxidized  two  carbon  atoms  at  a  time,  the  final  products  normally 
being  converted  completely  into  carbon  dioxide  and  water.  The 
acetoacetic  acid  CHg-^CO— CH^— COOH,  /?-oxybutyric  CH3— 
CHOH— CH2— COOH  and  acetone  which  appear  in  the  urine  in 
the  advanced  stages  of  diabetes,  when  the  organism  is  so  severely 
affected  that  its  powers  of  oxidation  are  impaired  are  believed  to 
be  largely  a  product  of  the  incomplete  oxidation  of  fatty  acids, 
though  acetoacetic  has  been  shown  to  arise  also  from  certain  of 
the  amino  acids. 

Metabolism  of  Inorganic  Material. — The  metabolism  of  inor- 
ganic material  assumes  an  aspect  somewhat  different  from  that 
of  the  organic  substances,  for  the  reason  that  inorganic  sub- 


METABOLISM  191 

stances  are  not  disintegrated  or  "burned"  in  the  organism,  and 
thus  are  not  sources  of  energy.  There  is,  of  course,  more  or  less 
interchange  of  radicles,  and  to  a  certain,  extent  inorganic  sub- 
stances are  built  into  compounds  of  organic  nature.  But  quite 
aside  from  this,  inorganic  materials  play  a  very  important  part 
in  metabolism.  Numerous  chemical  reactions  in  the  body  are 
controlled,  or  influenced  by  salts.  The  irritability  of  muscle  and 
nerve  is  greatly  affected  by  the  kind  and  amount  of  salts,  pres- 
ent. The  clotting  of  blood  and  of  milk  both  are  dependent  upon 
the  presence  of  calcium.  The  general  osmotic  equilibrium  of 
tissues  and  fluids  depends  in  large  measure  upon  salts.  Even 
the  development  of  the  unfertilized  eggs  of  some  animals  may 
be  stimulated  by  certain  salts.  Evidently  the  inorganic 
materials  of  the  body  have  far  more  extended  importance  than 
merely  to  form  an  inert  framework  to  support  and  protect  the 
soft  organs  and  tissues. 

Energy  Exchange. — A  very  important  phase  of  metabolism 
is  concerned  with  the  body's  energy  requirements.  Very  amus- 
ing ideas  on  this  subject  prevailed  up  to  a  century  and  a  half 
ago.  The  foundations  of  present  day  knowledge  were  laid  by 
the  great  Frenchman,  Lavoisier,  who  demonstrated  that  there 
was  something  in  common  between  the  burning  of  a  candle  and 
the  breathing  of  an  animal.  Mice  died  and  candles  went  out  if 
placed  under  a  bell  jar,  and  either  one  shortened  the  period  of 
a  subsequent  mouse  or  candle.  He  decided  that  the  burning  of 
the  candle  consisted  in  a  combining  of  the  carbon  of  the  candle 
with  a  substance  in  the  air  which  he  called  ' '  oxygen, ' '  and  that 
in  animals  a  similar  process  took  place,  producing  the  heat  of 
the  body.  By  measuring  the  amount  of  ice  melted  in  a  given 
time  by  the  heat  from  an  animal,  and  comparing  the  result  with 
the  amount  of  carbon  dioxide  produced  b}^  the  animal  in  a  period 
of  similar  length,  he  obtained  results  Avhich  indicated  that  at 
least  96%  of  the  heat  produced  by  th'e  animal  could  be  accounted 
for  by  the  oxidation  of  carbon  to  COg.  He  further  showed  that 
oxygen  also  was  used  up  in  combining  with  hydrogen  to  form 
water.     He  also  arrived  at  the  result  that  more  heat  was  pro- 


192  PHYSIOLOGICAL    CHEMISTRY 

duced  when  the  animal  was  subjected  to  cold  than  otherwise, 
and  that  digestion  and  work  increased  the  heat  output.  Un- 
fortunately, Lavoisier  lost  his  life  on  the  guillotine  during  the 
Reign  of  Terror,  and  his  brilliant  work  was  left  unfinished. 

The  way  had  been  opened,  however,  and  other  workers  took 
up  the  problems  which  the  brilliant  Lavoisier  had  opened.  The 
methods  and  ideas  of  Lavoisier  were  extended  and  im- 
proved. Dulong  and  Depretz,  Regnault  and  Reiset,  Rubner  and 
others  abroad,  and  Atwater,  Benedict,  Lusk,  du  Bois  and  others 
in  this  country  have  investigated  and  solved  many  of  the  prob- 
lems connected  with  energy  balance  and  energy  requirements. 
The  work  of  Atwater  in  this  country  has  been  of  particular 
service  from  the  point  of  technique  development.  Atwater,  in 
conjunction  with  Rosa  constructed  an  apparatus  known  as  a 
calorimeter.  This  contrivance  consists  of  an  insulated  chamber 
large  enough  for  a  man.  The  walls  are  so  constructed  that  no 
heat  is  lost  through  them.  The  heat  produced  by  the  occupant 
is  carried  off  by  water  circulating  through  a  cooling  coil.  The 
flow  of  water  and  its  temperature  at  entering  and  leaving  the 
chamber  can  be  accurately  measured,  and  thus  the  amount  of 
heat  carried  off  from  the  chamber  computed.  A  circulation  of 
air  is  provided,  and  from  this,  the  moisture  evaporated  from  the 
subject  is  extracted  and  measured,  and  the  heat  consumed  in 
its  vaporization  calculated.  A  third  factor  in  the  heat  measure- 
ment consists  in  observing  any  change  in  the  temperature  of  the 
patient  or  the  apparatus.  The  sum  of  these  three  factors  will 
give  the  total  heat  given  off  by  the  subject. 

The  circulating  air  is  in  a  closed  system.  The  carbon  dioxide 
is  removed  by  passing  the  air  through  an  absorption  apparatus, 
and  its  amount  can  be  determined  by  weighing.  Oxygen  is  sup- 
plied from  an  oxygen  retort,  so  that  the  amount  used  by  the 
subject  can  be  determined  accurately  by  the  loss  of  weight  of 
the  retort,  considered  in  connection  with  any  change  in  the 
composition  of  the  circulating  air. 

By  means  of  this  calorimeter,  accurate  measurements  may  be 
made  of  the  heat  produced  in  the  body,  and  the  amount  of  car- 


METABOLISM  193 

bon  dioxide  and  water  produced.  Results  of  the  most  extra- 
ordinary accuracy  have  been  obtained  by  Atwater,  Benedict, 
Lusk,  du  Bois,  and  others,  who  have  worked  with  apparatus 
constructed  on  this  general  principle. 

One  of  the  most  important  and  far  reaching  results  obtained 
in  this  way  is  that  the  law  of  conservation  of  energy  holds  for 
the  animal  body.  The  animal  body  can  neither  create  nor 
destroy  energy.  In  a  series  of  experiments  representing  an 
exchange  of  over  590,000  calories,  the  dilference  between  the 
theoretical  calculation  and  the  amount  actually  measured  was 
only  501  calories,  an  error  of  only  about  0.1%.  Thus  an  animal 
works  on  the  same  principle  as  an  engine,  a  candle  or  an  alcohol 
flame.  The  heat  which  it  produces  comes  from  the  oxidation  of 
organic  substances,  either  those  of  the  food,  or  those  of  the  body 
tissues. 

In  calculating  the  amount  of  heat  which  will  be  produced  in 
the  body  by  a  given  foodstuff,  it  must  be  borne  in  mind  that 
only  the  portion  of  the  food  which  is  absorbed  will  be  available 
for  burning  in  the  cells,  and  also  that  if  the  food  is  not  com- 
pletely burned  to  carbon  dioxide  and  water,  this  fact  must  be 
taken  into  account.  The  carbohydrates  ordinarily  are  com- 
pletely burned  to  COg  and  HoO,  as  are  also  the  fats.  The  pro- 
teins, however,  are  not  completely  oxidized,  for  uric  acid,  urea, 
and  other  substances  of  protein  origin  are  excreted  in  the  urine. 
The  figures  usually  used  for  the  average  energy  value  of  the 
three  foodstuffs  are  carbohydrates  4.1  large  calories  per  gram, 
proteins  4.1  and  fats  9.3.  It  will  be  seen  that  the  fats  yield  by 
far  the  most  heat  per  gram.  (It  will  be  recalled  that  a  large 
calorie  is  the  amount  of  heat  required  to  raise  1  kilo  of  water 
through  1°  C.  of  temperature, — usually  from  15°  to  16°  C.  or 
from  0°  to  1°  C.) 

Accurate  and  extended  studies  have  been  made  of  the  amount 
of  heat  produced  (and  thus  the  amount  of  fuel  required)  by 
individuals  in  health  and  in  various  diseases,  at  rest  and  during 
exercise,  waking  and  sleeping,  during  periods  of  mental  ease 
and  of  great  mental  exertion,  at  different  ages  from  infancy  to 


194  PHYSIOLOGICAL    CHEMISTRY 

old  age,  and  in  the  two  sexes.    It  will  be  possible  here  only  to 
summarize  briefly  some  of  these  important  findings. 

First,  the  different  foodstuffs  are  isodynamic  in  the  body,  that 
is,  the  body  can  employ  fats,  carbohydrates  or  proteins  as  fuel 
interchangeably,  and  with  no  loss  of  energy, — each  foodstuff 
furnishes  its  total  theoretical  amount  of  energy  when  it  is  oxi- 
dized in  the  body. 

The  total  amount  of  energy  required  by  an  individual  varies 
with  his  age,  state  of  health,  body  weight,  sex,  and  degree  of 
body  activity.  In  general,  the  energy  exchange  is  higher  in 
small  animals  than  in  larger  ones,  for  there  is  more  surface  per 
unit  of  weight  in  small  animals,  and  thus  the  loss  of  heat  is 
greater.  Metabolism  in  children  is  higher  than  in  adults,  partly 
for  the  foregoing  reason,  and  possibly  also  because  there  is 
greater  tissue  activity.  In  newborn  infants  during  the  first  few 
days  of  life,  metabolism  is  low.  In  a  fat  man  the  energy  ex- 
change per  kilo  body  weight  is  lower  than  in  a  thin  man,  as  the 
excess  weight  is  due  to  inert  fat  deposits  which  take  little  part 
in  active  metabolism.  Metabolism  is  lower  per  kilo  body  weight 
in  women  than  in  men. 

Increase  in  body  activity,  of  course,  increases  the  energy 
exchange.  More  fuel  is  burned,  and  more  heat  produced.  The 
average  city  adult  man  requires  from  2,500  to  3,000  calories  per 
day.  If  such  a  person  remains  in  bed  and  inactive,  his  energy 
requirement  sinks  to  perhaps  1,700  calories.  Physical  exercise 
and  exposure  to  cold  greatly  increase  the  energy  requirement, 
which  may  run  up  to  4,000  or  even  5,000  calories.  The  extreme 
limit  recorded  is  the  case  of  an  endurance  bicycle  rider  who  used 
up  10,000  calories  in  a  day. 

An  interesting  fact  is  that  the  mere  taking  of  food  will  mate- 
rially increase  the  heat  production  in  the  body.  It  was  suggested 
that  this  was  due  to  the  work  of  the  digestive  glands  and  organs, 
but  this  has  been  shown  to  be  incorrect ;  the  taking  of  salts  which 
cause  intense  muscular  activity  of  the  intestine  resulted  in  no 
such  increase.  This  effect  of  food  substances  is  spoken  of  as 
the  Specific  Dynamic  action  of  the  foods.     Proteins  cause  the 


METABOLISM  195 

greatest  rise  in  heat  production.  The  effect  in  the  case  of  pro- 
teins is  believed  to  be  due  to  a  stimulation  of  the  cells  of  the 
tissues  to  greater  activity,  the  stimulation  being  produced  by  the 
decomposition  fragments  of  protein  in  the  blood.  In  the  case 
of  the  carbohydrates  and  probably  also  of  the  fats,  the  effect  is 
believed  to  be  due  to  the  "mass  action"  of  the  fragments  of 
decomposition  and  not  to  a  direct  stimulating  action.  Since 
proteins  produce  the  greatest  increase  in  heat  production,  they 
should  be  eaten  sparingly  in  hot  weather. 

Curiously  enough,  increased  mental  activity  does  not  increase 
the  heat  produced  by  the  body.  This  might  be  interpreted  to 
mean  that  mental  activity  uses  up  no  fuel,  or  an  inappreciable 
amount,  but  it  is  more  nearly  accurate  to  state  that  great  mental 
activity  such  as  studying  for  an  examination  produces  no  more 
heat  than  the  mental  activity  of  our  ordinary  and  most  indolent 
mental  processes. 

During  sleep  metabolism  is  low,  but  this  is  due  probably  to 
lessened  body  activity  rather  than  to  any  inherent  differences 
of  metabolism. 

The  variations  of  heat  production  in  disease  have  been  care- 
fully studied  by  Benedict,  Lusk,  du  Bois  and  others.  In  typhoid 
and  exopthalmic  goitre  there  is  a  great  increase.  In  diabetes 
the  increase  is  slight. 

Utilization  of  Alcohol  by  the  Body. — A  problem  of  the  great- 
est importance  has  been  to  determine  the  position  of  alcohol  in 
metabolism.  If  burned  in  a  calorimeter,  alcohol  yields  over  7 
calories  per  gram,  and  thus  has  a  very  high  fuel  value.  A  long 
and  careful  study  of  the  effects  of  alcohol  on  the  body  has  been 
undertaken  by  Benedict.  In  the  body,  small  amounts  of  alcohol 
undoubtedly  are  burned  as  fuel  by  the  tissues.  The  effects  of 
alcohol  are  those  of  a  depressant  rather  than  of  astimulant, 
apparent  stimulation  being  in  reality  a  depression  of  inhibiting 
influences.  Although  alcohol  undoubtedly  is  burned  as  fuel  by 
the  body,  still  it  is  a  generally  accepted  fact  that  its  poisonous 
effect  on  the  cells  more  than  counterbalances  any  beneficial  effect 
resulting  from  its  high  fuel  value. 


196  PHYSIOLOGICAL   CHEMISTRY 

Starvation. — If  an  animal  is  deprived  of  food  it  will  live  for 
some  time,  drawing  upon  its  own  tissues  for  the  essential  ma- 
terials for  fuel  and  body  maintenance.  The  length  of  time  an 
animal  will  survive  without  food  varies  greatly  with  the  size 
and  condition  of  the  animal,  in  general  a  large  or  a  fat  animal 
will  survive  longer.  It  depends  also  much  on  external  condi- 
tions. If  exposed  to  cold  or  great  exertions  as  in  winter,  in 
shipwreck,  etc.,  life  may  be  lost  in  a  few  hours.  Otherwise,  fast- 
ing may  be  continued  for  even  a  month  or  longer  in  the  case  of 
a  man,  without  death  ensuing.  Children  die  much  sooner, 
usually  in  four  or  five  days.  Recently  the  case  of  a  dog  was 
reported  in  which  the  animal  survived  117  days  of  fast.  If 
water  is  withheld  as  well  as  food,  death  occurs  in  a  few  days' 
time. 

The  study  of  metabolism  during  fasting  furnishes  interesting 
information  about  the  chemical  processes  going  on  in  the  body. 
Benedict  has  published  an  exhaustive  study  of  a  man  fasting  31 
days. 

During  fasting  there  is  of  ocurse  continuous  loss  of  body 
weight.  Reserve  stores  of  glycogen  and  fat  are  called  upon,  but 
there  also  is  a  continuous  excretion  of  nitrogenous  material  in 
the  urine,  showing  that  some  protein  is  continually  broken  down. 
At  an  earlier  point  we  have  noted  the  appearance  of  creatine  in 
starvation,  replacing  a  portion  of  the  normal  creatinine.  A  most 
interesting  fact  is  that  all  tissues  and  organs  do  not  lose  weight 
in  like  amount  as  starvation  proceeds.  Organs  of  vital  impor- 
tance such  as  the  heart,  the  brain  and  nerves  are  preserved  prac- 
tically without  loss  of  weight,  whereas  the  skeletal  muscles,  the 
liver  (loses  glycogen)  and  adipose  tissue  lose  a  very  considerable 
portion  of  their  weight.  The  body  tries  to  save  the  most  vitally 
important  organs,  and  does  so  at  the  expense  of  less  indispen- 
sable tissues,  which  are  called  upon  to  furnish  fuel  and  undoubt- 
edly also  repair  material  for  the  vital  parts. 

The  nitrogen  excretion  of  a  fasting  animal  gradually  de- 
creases as  time  goes  on,  probably  as  a  result  of  the  decreased 
amount  of  protein  tissue  in  the  body.    Shortly  before  the  death 


•  METABOLISM  197 

of  the  animal  the  nitrogen  excretion  rises  sharply.  This  is  known 
as  the  pre-mortal  rise.  It  appears  as  if  the  organism  gives  up 
the  struggle  to  protect  its  tissues  from  destruction  and  that  the 
overtaxed  organism  begins  to  give  way.  After  a  prolonged  fast, 
the  total  nitrogen  excretion  in  man  falls  to  the  neighborhood  of 
about  2  grams  per  day.  This  appears  to  be  the  minimum,  and 
has  been  taken  by  some  to  represent  the  actual  necessary  wear 
and  tear  of  the  protein  tissues  of  the  body.  Possibly,  however,  it 
is  necessary  for  the  body  to  break  down  the  protein  which  this 
represents  in  order  to  obtain  some  particular  amino  acid  or 
acids  needed  in  the  synthesis  of  some  absolutely  essential  prod- 
uct of  internal  secretion. 

Several  cases  are  on  record,  and  the  author  himself  has  known 
individuals  who  have  derived  great  benefit  from  an  occasional 
wisely  timed  fast.  The  digestive  and  excretory  organs  are  given 
a  rest,  the  tissues  are  cleared  of  all  unnecessary  stored  up  ma- 
terial, and  the  body  undergoes  a  thorough  housecleaning. 

Unknown  Food  Constituents. — Vitamines. — Until  recently  it 
was  believed  that  if  an  individual  were  supplied  with  enough 
protein,  the  necessary  salts,  and  amounts  of  carbohydrates  and 
fats  sufficient  to  make  up  the  total  of  energy  required,  that  his 
diet  was  adequately  regulated.  Recent  developments  have 
shown  that  this  list  is  incomplete,  and  that  certain  substances 
hitherto  undreamed  of,  are  absolutely  essential  to  the  health  and 
even  the  life  of  the  individual.  The  chemical  nature  of  these 
substances  is  still  unknown,  but  it  appears  that  there  are  at 
least  two  and  possibly  more  of  them. 

The  discovery  of  the  existence  of  these  substances  was  inti- 
mately connected  with  a  disease  knowTi  as  beri-beri  prevalent  in 
oriental  countries  where  the  natives  live  on  a  diet  consisting 
mainly  of  polished  rice.  On  such  a  diet,  extensive  disturbances 
of  the  nervous  system  develop,  resulting  in  weakness,  paralysis 
and  ultimate  death.  Feeding  the  outside  layer  of  rice,  ordi- 
narily removed  in  polishing,  causes  immediate  relief  and  speedy 
recovery.  A  substance  evidently  is  present  in  the  outer  layer 
of  rice,  without  which  an  animal  on  a  polished  rice  diet  devel- 


198  PHYSIOLOGICAL   CHEMISTRY 

ops  beri-beri,  A  similar  condition  appears  in  chickens  on  a 
rice  diet,  and  extensive  studies  have  been  carried  out  to  ascer- 
tain the  nature  of  the  curative  substance  and  of  the  disease. 
Funk  has  called  the  unknown  material  ''vitamine, "  a  name  to 
which  there  are  some  objections,  and  believes  that  the  substance 
contains  nitrogen.  MeCoUum,  and  Osborne  and  Mendel  recog- 
nize two  substances,  one  of  them  present  in  butter  fat,  and  dif- 
fering from  the  substance  in  rice  polishings  by  containing  no 
nitrogen.  The  substance  in  butter  fat  appears  to  be  essential 
to  growth.  MeCollum  has  adopted  the  terminology  "Fat  Sol- 
uble A"  and  ''Water  Soluble  B"  for  the  two  materials.  Al- 
though much  labor  has  been  expended  on  the  problem,  the 
chemical  nature  of  these  various  important  materials  still  is 
unknown.  A  great  variety  of  compounds  have  been  studied, 
and  shown  not  to  be  the  desired  unknown.  Undoubtedly  the 
problem  will  be  solved  in  the  not  distant  future. 

The  investigation  of  these  unknown,  but  essential  dietary 
constituents  is  made  difficult  by  the  fact  that  they  are  present 
in  the  foods,  and  are  required  by  the  body  in  extremely  minute 
amounts,  a  few  miligrams  being  sufficient  to  supply  all  the 
body's  daily  needs. 

Various  diseases  other  than  beri-beri,  such  as  scurvy  and 
pellagra,  also  have  been  considered  by  some  to  be  due  to  lack 
of  some  such  unknown  substances.  They  have  been  classed  as 
' '  deficiency  diseases. ' '  Evidence  is  not  conclusive  on  this  point, 
and  it  is  perhaps  wiser  to  await  further  developments  before 
drawing  conclusions.  Recent  unpublished  results  of  MeCollum 
seem  to  indicate  that  scurvy  does  not  belong  in  this  class. 

Fortunately,  the  unknown  dietary  substances  are  fairly  widely 
distributed  in  ordinary  foodstuffs.  Butter  fat,  milk,  meat, 
yeast,  orange  juice,  fresh  vegetables  and  other  common  articles 
of  diet  contain  one  or  more  of  the  unknown  substances  which 
the  body  needs.  Just  why  the  body  needs  these  materials  and 
the  functions  which  they  perform  are  matters  as  yet  obscure. 
Possibly  they  stimulate  or  regulate  the  functioning  of  certain 
tissues  or  cells,  possibly  they  are  used  as  materials  for  the  con- 


METABOLISM  199 

struction  of  certain  products  of  internal  secretion  which  are 
required  in  the  processes  of  metabolism.  The  riddle  is  still 
unsolved,  but  nevertheless,  we  are  closer  to  its  solution  than 
were  our  ancestors,  who  did  not  dream  of  the  existence  of  these 
vitally  important  compounds. 

Body  Temperature. — In  warm  blooded  animals  body  temper- 
ature must  be  maintained  at  a  constant  level,  regardless  of 
external  conditions.  Fuel  is  burned  in  the  body,  and  heat  pro- 
duced. In  lower  animals,  a  fall  in  the  external  temperature 
seems  directly  to  stimulate  increased  heat  production  in  the 
body.  In  man  such  a  direct  result  appears  to  be  .wanting,  but 
the  same  end  is  accomplished  indirectly  by  stimulation  of  shiver- 
ing or  increased  voluntary  activity.  More  fuel  is  burned,  and 
more  heat  produced. 

It  appears  that  the  heat  situation  in  the  body  is  presided  over 
by  a  center,  possibly  two  centers  in  the  medulla.  One  of  these 
centers  presides  over  heat  production,  the  other  over  a  balance 
between  heat  production  and  heat  loss.  Heat  loss  is  accom- 
plished by  increased  excretion  of  sweat,  which  evaporates  from 
the  skin  and  thus  removes  excess  heat.  Sweating  may  occur 
even  in  cold  weather  during  vigorous  exercise,  when  the  heat 
production  is  greatly  increased.  If  evaporation  from  the  sur- 
face of  the  body  is  prevented,  either  by  coating  the  body  with 
some  substance,  or  if  the  surrounding  air  is  saturated  with 
moisture,  great  discomfort  is  experienced.  The  temperature  of 
the  body  rises,  and  death  will  result  if  the  condition  is  not 
relieved. 

Influence  of  Organs  of  Internal  Secretion. — The  metabolism 
of  the  body  as  a  whole,  and  of  specific  organs  or  tissues  is  pro- 
foundly influenced  by  products  poured  out  into  the  blood  stream 
by  certain  glands  and  other  tissues.  Since  these  products  are 
delivered  into  the  blood  stream  they  are  called  internal  secre- 
tions. We  already  have  considered  the  internal  secretion  of  the 
adrenals,  adrenaline,  in  its  effect  upon  the  control  of  liver 
glycogen,  the  internal  secretion  of  the  pancreas  and  its  impor- 
tance for  the  utilization  of  sugar  by  the  muscles,  the  hormones 


200  PHYSIOLOGICAL    CHEMISTRY 

secreted  by  the  stomach  and  intestinal  walls,  and  their  impor- 
tance in  inducing  the  flow  of  digestive  juices.  To  this  list  many- 
more  examples  might  be  added.  Thus  the  thyroid  gland  is 
known  to  have  a  profound  influence  on  general  metabolism.  If 
the  gland  atrophies,  or  is  removed  in  a  young  individual, 
growth  is  retarded  and  dwarfism  results.  Mental  development 
also  is  checked.  The  condition  is  known  as  cretinism,  and  it  may 
be  improved  by  feeding  thyroid.  In  adults,  removal  or  atrophy 
of  the  gland  results  in  thickening  of  the  skin,  and  coarseness 
of  the  hair,  the  temperature  is  below  normal  and  general  depres- 
sion of  metabolism  is  observed.  If  the  thyroid  is  too  active  or 
too  large,  goitre  results,  with  a  general  quickening  of  metab- 
olism and  increased  nervous  irritability.  These  and  other  facts 
are  interpreted  to  mean  that  the  thyroid  pours  out  a  substance 
into  the  blood  which  has  a  profound  effect  on  general  metab- 
olism, and  influences  the  metabolism  of  the  bones,  the  nerves 
and  the  skin.  This  substance  undoubtedly  contains  iodine.  It 
is  being  studied  by  Kendall.  The  parathyroids  undoubtedly  also 
have  important  functions,  but  they  are  not  so  well  understood. 
Careful  removal  of  these  organs  causes  symptoms  of  poisoning, 
but  the  cause  is  still  obscure. 

In  addition  to  the  action  of  adrenaline  above  mentioned,  this 
secretion  of  the  suprarenal  glands  increases  vasomotor  tone  and 
has  other  less  understood  functions.  Even  if  adrenaline  is 
injected  into  an  animal  from  which  the  suprarenals  have  been 
removed,  the  animal  dies,  but  the  cause  of  its  death  is  unknown. 

The  sexual  glands  also  markedly  influence  metabolism.  Re- 
moval of  the  testes  in  immature  males,  a  process  known  as  cas- 
tration, results  in  the  failure  of  the  secondary  sexual  character- 
istics to  appear.  Also  the  corpora  lutea  are  believed  to  in- 
fluence the  growth  and  functioning  of  the  mammary  glands. 
Both  of  these  effects  are  considered  to  be  due  to  substances 
poured  out  into  the  blood  stream  from  these  organs. 

The  anterior  lobe  of  the  pituitary  body  is  known  to  have  a 
marked  influence  on  growth.  If  it  is  removed  from  a  young 
animal,  the  animal  fails  to  grow,  and  retains  its  infant  char- 


METABOLISM  201 

acteristics  such,  as  immature  intelligence,  infantile  sex  organs, 
etc.  If  there  is  an  excessive  development  of  this  portion  of  the 
hypophysis,  gigantism  results.  The  posterior  lobe  seems  to  have 
no  such  function.  The  only  symptoms  reported  after  its  removal 
are  an  increased  sexual  desire,  and  an  increased  flow  of  urine. 
The  thymus  and  pineal  glands  probably  have  functions  in 
connection  with  general  metabolism,  but  these  functions  as  yet 
are  obscure. 


PART  II 


LABORATORY  WORK 

Directions  for  laboratory  work  for  each  chapter  except  VI, 
X  and  XII  are  appended.  Lists  of  apparatus  and  materials 
needed  have  been  included  as  an  aid  to  the  teacher.  Directions 
for  making  up  the  necessary  quantitative  solutions  will  be 
found  at  the  end  of  the  volume. 

MATERIALS 

The  following  materials  should  be  available : 

1.  Cleaning  fluid — a  saturated  solution  of  potassium  bichromate  in  con- 
centrated sulphuric  acid. 

2.  Soap  solution. 

3.  Sand  or  damp  sawdust,  placed  for  instant  use  to  extinguish  small  fires. 

4.  A  blanket  or  woolen  rug  is  a  valuable  aid  in  case  a  student's  clothing 
catches  fire. 

5.  A  supply  of  sodium  bicarbonate  solution  should  be  kept  on  hand  for 
use  if  a  student  gets  acid  in  his  eyes.  "Wash  under  running  water  and  pour 
on  sodium  bicarbonate  solution. 

6.  Boric  acid  solution  should  be  kept  for  use  if  a  student  gets  alkali  in 
his  eyes. 

The  following  list  includes  the  apparatus  desirable  for  the 
equipment  of  the  desk  of  each  student.  It  is  supplemented 
occasionally  by  articles  obtained  temporarily  from  the  storeroom. 


4  Beakers,  assorted  sizes. 

1  Graduate,  10  c.c. 

1  Doz.  test  tubes. 

1  Graduate,  100  c.c. 

1  Erlenmeyer  flask,  250  c.c. 

1  1  c.c.  pipette,  graduated  in  1/100 

1  Erlenmeyer  flask,  500  c.c. 

c.c. 

2  Florentine  flasks,  500  c.c. 

1  5  c.c.  pipette. 

1  Florentine  flask,  1000  c.c. 

1  10  c.c.  pipette. 

1  Funnel,  large. 

1  20  c.c.  pipette. 

1  Funnel,  small. 

1  25  c.c.  pipette. 

202 


MATERIALS 


203 


Watch  glasses. 

Wash  bottle. 

Kjeldahl  flasks,  500  c.c. 

Woulff  bottle,  CaClj  tube  and 
glass  pearls. 

Kjeldahl  condenser  bulb  and  con- 
nections. 

Burette  and  clamp. 

Large   glass  stoppered  bottle. 

Stirring  rod. 

Porcelain  casserole. 

Porcelain  crucible  and  cover. 

Small  evaporating  dish. 

Large  evaporating  dish. 

Thermometer. 

I  Pkg.  small  filter  paper. 


14  Pkg.  large  filter  paper. 

Several  sheets  hard  filter  paper. 

2  Bottles  litmus  paper. 

1  Test  tube  holder. 

1  Test  tube  brush. 

1  Test  tube  rack. 

1  Clay  triangle. 

1  Wire  gauze. 

1  Iron  stand  and  ring. 

1  Tripod  with  2  rings. 

1  Bunsen  burner  and  tubing. 

1  Box  of  matches. 

1  Glass  slide. 

Key  to  locker. 

Several  small  labels. 


On  each  desk,  or  conveniently  placed  should  be  the  following 
reagents : 


1.  Acid,  acetic  glacial. 

2.  Acid,  acetic,  10%. 

3.  Acid,  hydrochloric,  cone. 

4.  Acid,   hydrochloric,   10%. 

5.  Acid,  nitric,  cone. 

6.  Acid,  nitric,  10%. 

7.  Acid,  sulphuric,  cone. 

8.  Acid,  sulphuric,  10%. 

9.  Acid,  picric,  sat'd  sol. 

10.  Alcohol,  95%,. 

11.  Ammonium  hydroxide,  10%. 

12.  Ammonium  molybdate,  10%. 

13.  Ammonium  oxalate,  2%. 

14.  Ammonium  phosphate,  2%. 

15.  Ammonium    sulphocyanate,    2%. 

16.  Barium  chloride,  5%. 


17.  Chloroform. 

18.  Copper  sulphate,  5%. 

19.  Ether. 

20.  Fehling,  Sol.  A. 

21.  Fehling,  Sol.  B. 

22.  Ferric  chloride,  2%. 

23.  Aqueous  iodine,   1%. 

24.  Mercuric  chloride  sat. 

25.  Millons  reagent. 

26.  Potassium  ferrocyanide,  2%. 

27.  Silver  nitrate,  2%. 

28.  Sodium  carbonate,  2%. 

29.  Sat.  sodium  chloride. 

30.  Sodium  chloride,   10%. 

31.  Sodium  hydroxide,  sat. 

32.  Sodium  hydroxide,  10%. 


The  following  lists  cover  the  special  materials  and  apparatus 
needed  for  the  work  in  the  laboratory  chapters: 

Chapter  IL 

Solids.  Liquids. 

Meat    (hamburger    steak)     1    lb.  Lead   acetate  sol. 

enough  for  25  students.  Blood  or  blood  serum — 20  c.c.  per 

Solid  casein.  man. 


204 


PHYSIOLOGICAL    CHEMISTRY 


Solids. 
Soda  lime. 
Fusion  mixture. 
Bone  ash. 
Ground  bone. 
Bismuth  subnitrate. 


Liquids. 

Potassium  liydroxide,   25%. 
Potassium   pyroantimonate   sol. 
Sodium  cobaltinitrite   sol. 


Chapter  III. 


Solids. 
Dextrose. 

Yeast — 1  cake  for  25  students. 
Cane  sugar. 


for  each  student. 


Maltose. 

Lactose. 

Potatoes — 1 

Starch. 

Dextrin. 

Orcin. 

Phlorglucin. 


Special  Apparatus. 
Microscope. 

Polariscope  and  its  accessories. 
Fermentation  tube. 
Incubator. 
Steam  bath. 

Knives  for  scraping  potato. 
Cheese  cloth  for  straining. 


Solutions. 
Dextrose,  2%. 
Levulose,  2%. 
Galactose,  1%. 
Arabinose   (Hydrolize  gum  arable 

with  2%  H,SOJ. 
Cane  sugar,  2%. 
Maltose,  2%. 
Lactose,  2%. 
Glycogen. 
Barfoeds  sol. 
Nylanders. 
Phenylhydrazine. 
15%  alcoholic   cc    naphthul. 
Tannic  acid  sol. 
Amyl  alcohol. 


Chapter  IV. 


Solids. 
Lard. 

Boric  acid  or  KHSO^. 
Fusion  mixture. 


Liquids. 

Olive   (or  cottonseed)    oil. 

Soap  solution. 

Albumin  solution. 

Gum  arabic  sol. 

Lymph   (few  drops  if  available). 

Alcoholic  NaOH,  75  c.c.  per  man. 

Glycerine. 

Calcium  chloride  sol. 

Egg  yolk  or  its  ether  extract. 

Acetone,  40  c.c.  per  man. 

Phenolphthalein,    1%    alcoholic. 


MATERIALS 


205 


Chapter  V. 


Solids. 

For  Simple  Frotrivfi. 

Casein. 

Magnesium  sulphate. 

Ammonium  sulphate. 

Ego-   albumin. 

Fibrin. 

Meat  (muscle). 

Flour,  50  grams  per  man. 

Horn. 

Tendo  Achilles. 

Gelatine. 

Ligamentum  nuclise. 
For  Conjugated  Proteins. 

Solid  benzidene. 

Carrots — raw  and  boiled. 

Beef  pancreas. 
For  Derived  Proteins. 

Witte  's  peptone. 

Armour's  peptone. 

Barium  carbonate. 

Wool. 

Charcoal. 

Solid  sodium  acetate. 

Congo  paper. 

Fusion  mixture. 

Special  Apparatus. 
Spectroscopes. 

Flat  sided  cells  for  spectroscopic 
observations. 


Liquids. 

For  Simple  Proteins. 
Egg    albumin    sol. 
Phenol,   1%  sol. 
Glyoxylic  acid  sol. 
Alcoholic    °'-     naphthol,  15%. 
Potassium  mercuric  iodide  sol., 

2%. 
Acid  phosphomolybdic,  2%. 
Acid  phosphotungstic,  2%. 
Acid  trichloracetic,  2%. 
Tannic  acid. 
Lead  acetate  sol. 
Ammonium  sulphate,  sat. 
Blood  serum. 
Egg   white,   undiluted,   5   c.c.   per 

man. 
Blood  plasma,  20  c.c.  per  man. 
Pepsin  sol.,  1%  in  0.2%  HCl. 
Pancreatin   sol.,   1%    aqueous. 
Lime  water,  sat. 
For  Conjugated  Proteins. 
Defibrinated  blood. 
Stokes'  reagent. 
CO-hemoglobin,  aerated. 
Fresh     potassium     ferricyanide 

sol. 
Defibrinated   rat  or   guinea  pig 

blood. 
Hydrogen  peroxide. 
Guaiac. 

Corpuscles,  washed  in  physiol.  sa- 
line. 
Milk,  30  c.c. 
Lead  acetate  sol. 
Egg  yolk  extracted  with  eiher. 
Egg  white  diluted  1  to  10. 
Derived  Proteins. 

Egg  white  undiluted  10  c.c. 
Meat  digested  with  pancreatin. 


206 


PHYSIOLOGICAL    CHEMISTRY 


Solids. 
Paraffin. 
Starch. 
Freezing  mixture  (ice  and  salt) 


Chapter  VII. 

Liquids. 


Phenolphthalein,     1%     alcoholic 

sol. 
Methyl  orange,   1%   aqueous. 
Cane  sugar  sol. 


Chapter  VIII. 


Solids. 
Pig's  stomach. 
Mett's  tubes. 
Thread. 

Rennin  tablets. 
Capsules,  0.1  gram  CHI3. 
Capsules,  0.2  gram  KI. 
Capsules,  1.0  gram  salol. 
Starch  paper. 
Solid  ammonium  sulphate. 


Solids. 
Mett's  tubes. 
Pancreatin  powder. 
Cane  sugar. 
Charcoal. 

Powdered  sulphur. 
Gall  stones. 


Solids. 

Sodium  carbonate. 
Barium  carbonate. 
Pure  oxalic  acid. 
Pure  sodium  bicarbonate. 
Potassium  sulphate. 


Special  Apparatus. 
Bottles   for   urine. 
Large  (2  liter)  cylinders. 
Urinometer. 
Weighing  bottles. 
Quantitative  balance. 


Liquids. 
Glycerine. 
Toepfer's  reagent. 
Guenzburg's  reagent. 
Congo  red. 
Phenol,  1%  sol. 
Lactic  acid,  1%. 
Milk. 

N/10  alkali. 
CaCL. 


Chapter  IX. 


Liquids. 

1%  aqueous  pancreatin  sol. 
Milk,  5  c.c.  per  man. 
Litmus  sol. 
Bile. 


Chapter  XI. 


Liquids. 

Urine,  2  or  3  liters  per  man. 

Albumin  urine. 

Sugar  urine. 

Acetone  urine. 

Urine,  a  24-hour  specimen. 

5%  Thymol  in  chloroform. 

Magnesia    mixture,    225    c.c. 

man. 
Potassium  permanganate — about  8 

or  10%  sol. 
Oxalic  acid,  about  5%. 
Bromine  water. 
Glycerol. 


per 


MATERIALS 


207 


Special  Apparatus. 

Fermentation  tubes. 

Polariscope. 

Esbach  tubes. 

100  c.c.  volumetric  flask. 

500  c.c.  volumetric  flask. 

Duboscq  colorimeter. 

Distilling  apparatus. 

Cylinders,  bottles,  Folin  absorp- 
tion tubes  for  ammonia  and 
urea  determinations. 

Compressed  air  (or  a  water 
pump). 


Liquids. 

Apparatus  for  producing  H,  S. 

Sodium  nitroprusside  sol. 
(fresh). 

Methyl  orange,  1%  aqueous. 

Calcium   chloride   sol. 

Lead  acetate  sol. 

Alizarine  red,  1%  aqueous. 

1%  Potassium  dihydrogen  phos- 
phate, 30  c.c.  per  man. 

Neutralized  potassium  oxalate 
sol.   (saturated). 

Sodium  hydroxide,  saturated. 

Kerosene. 

Formalin,  diluted,  1-4  and  neu- 
tralized with  N/10  NaOH 
(phenolphthalein) . 

Folin-Schaffer  reagent. 

10%  Ammonium  sulphate. 

N/20  Potassium  permanganate. 

N/2   Potassium  bichromate. 

Standard  silver  nitrate. 

Standard  ammonium  sulphocyan- 
ate. 

Ferric  ammonium  sulphate,  sat'd 
(iron  alum). 

Standard  barium  chloride. 

Special  sodium  acetate. 

Standard  uranium  acetate. 

Esbach 's  reagent. 

Quantitative,  Fehling  's. 

Benedict's  sol. 


CHAPTER  I 

GENERAL  LABORATORY  INSTRUCTIONS 

Perform  all  experiments  with  care.  Always  read  through 
the  directions  before  beginning  work.  Record  results  imme- 
diately after  they  have  been  observed.  Do  not  wait  until  a  later 
time  and  then  trust  to  your  memory. 

Be  neat  and  careful  in  your  work. 

Always  return  reagent  bottles  to  their  proper  places  imme- 
diately after  using.  Keep  your  desk  and  apparatus  clean  and  in 
good  order.  After  being  used,  apparatus  should  be  thoroughly 
cleaned  before  it  is  put  away.  Wash  it  with  tap  water,  using  a 
brush,  and  finally  rinse  with  distilled  water.  Greasy  materials 
can  be  removed  with  warm  water  and  soap.  If  these  means  are 
not  effective,  use  cleaning  fluid  (potassium  bichromate  and  con- 
centrated sulphuric  acid).  Do  not  throw  the  cleaning  fluid 
away.  Return  it  to  the  vessel  from  which  it  was  taken  as  it  can 
be  used  again.  Glassware  cleaned  with  cleaning  fluid  should  be 
rinsed  thoroughly  with  tap  water  and  flnally  with  distilled 
water. 

Do  not  put  a  pipette  or  dropper  into  any  reagent  bottle.  If 
a  reagent  must  be  measured  with  a  pipette,  pour  out  a  small 
amount  into  a  clean  dry  beaker,  or  test  tube,  and  take  up  with 
a  pipette  from  this  vessel.  If  more  of  a  given  laboratory  reagent 
has  been  taken  from  a  bottle  than  is  needed,  do  not  return  the 
excess  to  the  original  bottle.  Be  careful  not  to  pour  out  more  of 
the  reagent  than  you  require,  but  if  excess  has  been  taken  it 
should  be  thrown  away. 

In  using  alcoJiol,  etlier,  acetone,  benzol,  etc.,  make  sure  tJiat 
no  fire  is  near.  Ether  vapor  is  especially  dangerous,  as  it  may 
flow  along  the  desk  top  for  some  distance  and  ignite  at  a  neigh- 
boring Bunsen  burner.    Small  desk  fires  often  may  be  put  out  by 

208 


GENERAL  LABORATORY  INSTRUCTIONS  209 

throwing  on  sand,  or  moist  sawdust,  which  should  be  kept  in 
boxes  throughout  the  laboratory.  It  is  an  excellent  plan  to  keep 
a  woolen  blanket  or  rug  in  the  laboratory.  In  case  a  student's 
clothing  has  taken  fire  he  may  be  Avrapped  in  the  blanket  and 
the  flames  extinguished.  Solutions  of  sodium  bicarbonate  and  of 
boric  acid  should  also  be  kept  in  the  laboratory  for  use  if  acid 
or  alkali  is  spattered  into  the  eyes  or  face. 

In  testing  for  substances  present  in  small  amounts,  make  sure 
that  both  the  liquid  to  be  tested  and  the  precipitating  reagent 
are  perfectly  clear,  filtering  them  repeatedly  if  necessary.  If 
uncertain  as  to  the  presence  of  a  precipitate,  compare  wdth  the 
original  solution. 

As  many  of  the  chemical  reactions  in  physiological  chemistry 
are  extremely  complex,  the  student  need  write  equations  only 
when  they  are  specifically  called  for  in  the  notes. 


CHAPTER  II 

DETECTION  OF  THE  ELEMENTS  AND  OF 
INORGANIC  SALTS 

The  body  tissues,  foodstuffs,  etc.,  are  made  up  of  a  large 
number  of  elements  which  are  seldom  free,  but  usually  com- 
bined in  the  form  of  organic  substances  or  inorganic  salts.  The 
latter  are  often  in  more  or  less  firm  combination  with  the  or- 
ganic constituents  of  the  tissues,.  The  inorganic  materials  often, 
but  not  always,  can  be  extracted  from  tissue  material  with  water 
or  dilute  acid.  To  detect  the  elements  in  organic  substances  it 
is  usually  necessary  to  destroy  the  organic  material  by  fusing 
it  with  an  oxidizing  agent.  Metals  or  salts  making  up  part  of 
the  compound  are  set  free  in  this  manner  and  can  be  detected  by 
the  usual  qualitative  tests.  The  following  experiments  will 
demonstrate  the  presence  of  the  various  elements  and  salts 
which  are  found  most  frequently  in  the  body  or  in  food 
materials. 

1.  1,  Carbon. — Place  a  small  quantity  of  sugar,  fat,  dry 
casein  or  meat  which  has  been  thoroughly  dried  and  ground  up 
in  a  test  tube  and  heat  in  the  Bunsen  flame.  The  material  is 
decomposed,  leaving  a  black  residue  of  carbon. 

2.  Hydrogen. — Observe  the  liquid  which  condenses  on  the 
inside  of  the  test  tube.  This  is  water.  Since  the  original  mate- 
rial was  dry,  this  water  must  have  been  formed  from  the 
hydrogen  in  the  substance,  and  oxygen  from  the  substance  itself 
or  the  air. 

3.  Oxygen. — There  is  no  simple  method  for  detecting  the 
presence  of  oxygen  in  combination.  In  analysis,  the  sum  of  all 
the  other  constituents  present  in  a  given  substance  is  subtracted 
from  the  weight  of  material  used.  The  difference  is  taken  as  the 
amount  of  oxygen  in  the  compound. 

210 


DETECTION   OF   ELEMENTS   AND    INORGANIC    SALTS  211 

4.  Nitrogen. — Mix  a  small  portion  of  casein  or  meat  with 
soda  lime  in  a  test  tube  and  warm  gently  in  the  flame.  (Soda 
lime  is  prepared  by  mixing  solid  sodium  hydroxide  and  calcium 
hydroxide  and  heating  the  mixture.)  Nitrogen  is  liberated  in  the 
form  of  ammonia.  To  detect  this  compound,  moisten  a  piece  of 
litmus  paper  with  distilled  water  and  hold  it  in  the  mouth  of  the 
tube.    It  should  turn  blue. 

5,  Sulphur. — Sulphur  may  be  present  as  "loosely  combined," 
that  is,  unoxidized  sulphur,  or  in  oxidized  form  as  sulphate. 

a.  To  detect  loosely  combined  sulphur  suspend  a  small  amount 
of  casein  in  water,  add  sodium  hydrate  and  a  few  drops  of  lead 
acetate  and  boil.  "Loosely  combined"  sulphur  is  split  off,  and 
combines  with  the  lead  to  form  lead  sulphide,  which  causes  the 
liquid  to  turn  dark  brown  or  black.  Add  sufficient  hydrochloric 
acid  to  make  the  solution  acid,  and  hold  in  the  mouth  of  the  test 
tube  a  strip  of  filter  paper  moistened  with  lead  acetate  solution. 
The  acid  liberates  hydrogen  sulphide  gas,  which  will  precipitate 
lead  sulphide  as  a  shiny  dark  material  on  the  filter  paper. 

b.  Oxidized  sulphur  is  usually  present  as  sulphate.  It  is 
detected  by  precipitation  as  barium  sulphate.  Sometimes  sulphate 
can  be  extracted  from  a  tissue  with  water  or  dilute  acid.  Some- 
times it  is  liberated  only  when  the  material  is  destroyed.  ( See  be- 
low under  the  analysis  of  special  tissues.) 

II.  Prepare  extracts  of  the  following  important  tissues  or 
fluids  and  test  them  for  inorganic  constituents.  Other  tissues 
may  be  substituted  if  desired.  Directions  for  the  specific  tests 
will  be  found  in  Section  III,  immediately  following  the  methods 
of  preparing  the  extracts. 

Prepare  each  material  for  analysis,  and  so  far  as  is  possible, 
complete  the  tests  upon  it  before  beginning  work  on  another 
material. 

1.  Preparation  of  Muscle  Extract. — Heat  100  c.c.  of  distilled 
water  to  boiling  and  add  a  small  teaspoonful  of  chopped  meat. 
Dilute  about  2  c.c.  of  10%  acetic  acid  with  about  18-20  c.c.  of 
distilled  water.  The  resulting  acid  will  be  about  1%.  Acidify 
the  water  and  meat  mixture  by  adding  2-3  drops  of  the  1% 


212  PHYSIOLOGICAL    CHEMISTRY 

acetic  acid  prepared,  and  boil  for  2-3  minutes,  stirring  with  a 
glass  rod.  If  the  liquid  does  not  clear,  add  3-4  drops  more  of 
the  acid  and  boil  again.  Observe  the  mixture  closely,  as  the 
liquid  may  be  quite  clear,  but  appear  cloudy  from  the  large  num- 
ber of  solid  particles  suspended  in  it.  Repeat  the  addition  of 
acid  and  boiling  if  necessary.  Caution:  Addition  of  too  much 
acid  will  cause  the  solution  to  be  muddy  in  appearance,  so  that 
it  will  not  filter  clear.  This  treatment  dissolves  out  a  large  part 
of  the  inorganic  salts,  and  coagulates  the  protein  material. 
Filter  the  liquid  from  the  solid  residue.  Test  the  filtrate  for 
inorganic  materials  as  described  below  in  Section  III. 

2.  Muscle  Residue. — The  coagulated  protein  material  and 
connective  tissue  also  contain  inorganic  substances,  but  in 
organic  combination.  To  detect  them  it  is  necessary  to  destroy 
the  organic  compounds  bj^  fusion  with  an  oxidizing  agent. 

Wash  the  coagulated  muscle  residue  with  a  little  hot  water 
and  dry  it  by  pressing  between  filter  papers.  Half  fill  a  crucible 
with  fusion  mixture.  This  is  a  basic  oxidizing  agent  which  con- 
tains KNO3  and  NaOH.  The  nitrate  furnishes  nascent  oxygen, 
and  the  sodium  hydrate  prevents  loss  by  volatilization  of  in- 
organic substances,  excepting  a  portion  of  the  carbonates. 

Support  the  crucible  in  a  clay  triangle  and. melt  the  fusion 
mixture  (hood)  by  heating  cautiously  with  the  Bunsen  flame. 
Add  small  portions  of  the  muscle  residue  until  the  charred  par- 
ticles do  not  disappear  on  heating  carefully.  When  this  point 
has  been  reached,  add  a  few  crystals  of  fusion  mixture,  and  heat 
until  colorless.  Allow  the  crucible  to  cool  and  dissolve  the 
residue  in  water  acidified  with  nitric  acid.  The  volume  should 
not  exceed  100  c.c.  Test  the  solution  with  litmus  (it  should  be 
acid)  and  make  phosphate,  sulphate  and  iron  tests  as  described 
below  in  Section  III.  What  do  you  conclude  as  to  the  forms  in 
which  phosphorus,  sulphur  and  iron  are  present  in  muscle 
tissue  ? 

3.  Blood  Serum. — Heat  100  c.c.  distilled  water  to  boiling, 
acidify  with  a  few  drops  of  1%  acetic  acid  and  pour  into  it 
slowly  20  c.c.  blood  serum  or  blood.    The  liquid  should  be  faintly 


DETECTION   OF   ELEMENTS   AND   INORGANIC   SALTS  213 

acid.  Test  with  litmus  and  add  more  of  the  acetic  acid  if  neces- 
sary to  produce  a  slightly  acid  reaction.  Boil  for  a  few  minutes. 
If  the  liquid  does  not  become  perfectly  clear,  add  a  few  drops 
more  of  the  acid  and  boil  again.  Avoid  excess  of  acid,  which 
will  cause  the  liquid  to  become  milky.  Filter  the  clear  liquid 
from  the  coagulated  proteins  and  test  it  for  inorganic  materials 
as  described  below  in  Section  III. 

4.  Bone. — Dissolve  a  small  spoonful  of  bone  ash  in  100  c.c. 
of  water  acidified  with  nitric  acid.  Is  a  gas  given  off?  If  so, 
what  substance  is  shown  to  be  present  in  bone?  (Remember  that 
in  preparing  the  bone  ash  much  CO2  may  have  been  driven  off 
by  heating.)  If  the  bone  ash  does  not  all  dissolve,  add  more 
nitric  acid,  or  filter  off  the  undissolved  ash.  Test  the  solution 
for  inorganic  materials  as  described  below  in  Section  III.  Be- 
fore testing  for  calcium,  neutralize  the  solution  with  ammonia 
(litmus)  and  reacidify  with  10%  acetic  acid,  as  calcium  oxalate 
is  soluble  in  nitric  acid.  In  case  the  phosphates  thrown  down 
by  the  ammonia  do  not  all  dissolve  on  the  addition  of  acetic 
acid,  the  solution  must  be  filtered  before  performing  the  calcium 
test. 

III.  Tests  for  Inorganic  Materials. — 

Use  about  3  c.c.  of  the  solutions  prepared  in  Section  II  for 
each  test.  Record  results  as  — ,  indicating  absence ;  -f ,  indicating 
traces;  and  -| — \-,  indicating  much.  If  the  liquid  only  becomes 
cloudy,  report  -j-.    If  there  is  a  distinct  precipitate,  report  -{ — 1— 

1.  Chlorides. — Acidify  with  about  %  volume  of  cone,  nitric 
acid  and  add  silver  nitrate.  The  presence  of  much  chloride  is 
indicated  by  a  heavy  white  precipitate.  If  only  traces  are  pres- 
ent, the  solution  becomes  cloudy  and  shows  a  bluish-white  opal- 
escence. Write  the  equation.  Confirm  the  presence  of  chloride 
by  adding  ammonium  hydrate  until  the  liquid  reacts  alkaline  to 
litmus.  On  adding  this  reagent,  any  precipitate  of  silver 
chloride  will  dissolve,  but  if  phosphates  are  present  in  quantity, 
they  will  be  thrown  down.  If  a  precipitate  forms  on  the  addi- 
tion of  ammonia,  filter,  and  in  the  filtrate  confirm  the  presence 


214  PHYSIOLOGICAL    CHEMISTRY 

of  chlorides  by  acidifying  and  reprecipitating  with  nitric  acid. 

2.  Sulphates. — ^Add  dilute  hydrochloric  acid  and  barium 
chloride.  Write  the  equation.  The  precipitate  is  barium  sul- 
phate. 

Note:  Sulphates  are  present  only  in  small  amounts  in 
most  body  tissues  and  fluids.  Unless  care  is  exercised,  a 
slight  precipitate  of  sulphates  will  be  overlooked.  The 
sulphate  test  should  be  observed  carefully  in  a  good  light, 
and  compared  with  the  original  solution  which  is  being 
tested  in  order  to  detect  a  possible  slight  precipitate. 

3.  Phosphates. — Add  1-2  c.c.  cone,  nitric  acid  and  about 
2-3  c.c.  ammonium  molybdate.  "Warm  carefully  until  just  too 
hot  to  be  held  in  the  hand,  and  allow  to  stand.  If  phosphates 
are  present,  a  yellow  crystalline  precipitate  of  ammonium  phos- 
pho-molybdate  will  gradually  settle  to  the  bottom  of  the  test 
tube.  The  precipitate  may  not  form  for  some  time,  but  its  ap- 
pearance often  nlay  be  hastened  by  rubbing  the  inside  of  the 
test  tube  gently  with  a  glass  rod.  If  the  precipitate  is  curdy, 
add  more  nitric  acid  and  boil  until  it  dissolves,  as  this  is  not 
the  phosphate  precipitate. 

4.  Carbonates. — The  carbonates  present  in  muscle  and  blood 
were  decom.posed  by  boiling  with  acid,  carbon  dioxide  being 
given  off.  Also  the  carbonates  in  bone  were  partly  destroyed  in 
the  preparation  of  bone  ash.  To  detect  carbonates  in  bone,  add 
2-5  c.c.  water  to  a  small  amount  of  ground  bone  and  add  a  few 
drops  of  concentrated  nitric  acid.  Observe  the  bubbles  of  COg. 
If  the  mouth  of  the  test  tube  is  held  to  the  ear,  the  effervescence 
may  be  plainly  heard. 

5.  Calcium. — To  10  c.c.  of  the  solution  add  about  2  c.c. 
ammonium  oxalate  and  allow  to  stand  for  15  minutes.  The 
precipitate  is  calcium  oxalate.  The  test  must  be  made  in  acetic 
acid  solution  as  nitric  acid  dissolves  calcium  oxalate.  Write  the 
equation.  Use  for  the  magnesium  test  the  portion  tested  for 
calcium. 

6.  Magnesium. — Filter  off  any  precipitate  of  calcium  oxalate 
in  the  liquid  tested  for  calcium.    If  the  filtrate  does  not  come 


DETECTION   OF   ELEMENTS   AND   INORGANIC    SALTS  215 

through  clear,  add  a  pinch  of  bismuth  subnitrate  and  refilter 
through  the  same  filter  paper.  Repeat  if  necessary  until  clear. 
The  bismuth  subnitrate,  which  is  extremely  insoluble,  closes  up 
the  larger  pores  of  the  filter  paper,  and  thus  holds  back  the  fine 
calcium  oxalate  crystals.  To  the  clear  filtrate  add  a  few  c.c.  of 
ammonium  oxalate  and  allow  to  stand  for  some  time,  preferably 
over  night,  to  make  sure  that  all  calcium  has  been  removed. 
After  complete  removal  of  the  calcium,  add  ammonium  hydrate 
and  ammonium  phosphate  to  the  clear  filtrate  and  allow  to  stand 
for  15  minutes.  The  precipitate  is  magnesium  ammonium  phos- 
phate. Write  the  equation.  Save  the  filtrate  for  the  sodium  and 
potassium  tests. 

7.  Iron. — Add  hydrochloric  acid  and  potassium  ferrocyanide 
to  the  liquid  to  be  tested.  In  the  presence  of  iron  ''Prussian 
blue"  is  formed.  A  small  amount  of  Prussian  blue  in  the  solu- 
tion may  result  only  in  a  green  color,  due  to  a  combination  of 
blue  with  the  yellow  already  present  in  the  solution.  A  green 
color  may  thus  be  taken  to  indicate  a  trace  of  iron.  If  more 
than  a  trace  of  iron  is  present  a  precipitate  will  form.  Write 
the  equation.  Compare  with  a  blank  test  on  the  reagents  used. 
If  iron  is  found,  remember  that  it  may  have  come  from  the 
hemoglobin  of  the  blood  present  in  traces  in  the  tissues. 

8.  Sodium. — Use  one-half  the  filtrate  from  the  magnesium 
test.  Make  sure  that  all  magnesium  has  been  removed  by  add- 
ing a  few  drops  of  ammonium  phosphate.  Divide  the  filtrate 
into  two  portions  and  reserve  one  for  the  potassium  test. 

To  test  for  sodium,  add  potassium  hydrate  a  few  drops  at  a 
time  and  warm  in  an  evaporating  dish  until  all  the  ammonia  has 
been  driven  off.  (Test  by  holding  over  the  dish  a  piece  of  red  or 
neutral  litmus  paper  moistened  with  distilled  water.)  Pour  into 
a  watch  glass  and  concentrate  on  the  steam  bath  to  about  14  c.c. 
There  should  be  no  solid  material  present  when  the  process  is 
stopped.  If  solid  material  has  appeared  in  the  liquid,  the  con- 
centration has  been  carried  too  far,  and  the  solid  should  be  dis- 
solved up  by  the  cautious  addition  of  water  drop  hy  drop,  stir- 
ring the  mixture  with  a  glass  rod.    Add  a  few  drops  of  potas- 


216  PHYSIOLOGICAL    CHEMISTRY 

slum  pyroantimonate  solution.  If  sodium  is  present  a  fine  white 
granular  precipitate  will  form  in  the  course  of  a- few  minutes 
and  stick  to  the  glass.  This  test  must  be  made  in  neutral  or 
alkaline  solution.  If  no  precipitate  is  visible,  observe  carefully 
in  a  good  light.  Rotate  the  watch  glass  carefully  so  that  small 
particles  of  precipitate  will  collect  in  the  center.  It  often  is  pos- 
sible to  hasten  the  formation  of  a  precipitate  by  rubbing  the  in- 
side of  the  containing  vessel  with  a  glass  rod. 

9.  Potassium. — From  the  portion  of  the  liquid  reserved  in  6  for 
the  potassium  test,  remove  ammonia  as  in  8,  but  use  sodium 
hydrate  instead  of  potassium  hydrate.  Add  glacial  acetic  acid 
until  the  liquid  is  acid  to  litmus.  Now  add  2-3  drops  of  sodium 
cobaltinitrite  solution.  In  the  presence  of  potassium  a  yellow 
crystalline  precipitate  forms  at  once. 


CHAPTER  III 

CARBOHYDEATBS 

Study  the  reactions  of  the  physiologically  important  carbohy- 
drates in  solutions  of  the  pure  substances,  as  this  method  is  more 
convenient  for  laboratory  piirposes  than  the  preparation  of  the 
materials  from  the  tissues  or  body  fluids  in  which  they  occur. 

I.  Monosaccharides 

/.  Hexoses. — This  group  includes  the  monosaccharides  of 
greatest  physiological  importance,  dextrose,  levulose  and  galac- 
tose. 

a  Dextrose. — (Glucose.) 

i.  Soluhility. — Test  the  solubility  of  dextrose  in  water,  95% 
alcohol  and  ether.  (Be  careful  of  fire.)  To  test  the  solubility  of 
a  substance  in  a  given  solvent,  take  a  small  amount  of  the  solid 
in  a  test  tube  and  to  it  add  a  few  cubic  centimeters  of  the  liquid. 
In  case  the  solid  does  not  completely  dissolve,  filter  and  test  the 
filtrate  for  the  substance  in  question  to  see  if  any  has  dissolved. 
This  may  be  done  either  by  evaporating  the  solution  to  dryness, 
or  by  chemical  means.  If  there  is  any  doubt  as  to  the  solubility 
of  dextrose  in  the  solvents  tried,  filter  and  evaporate  to  dryness 
on  the  steam  bath.  A  residue  indicates  that  a  portion  of  the 
material  has  dissolved. 

Test  the  solubility  of  dextrose  in  alcohol  diluted  with  an  equal 
volume  of  distilled  water.  Dextrose  is  soluble  in  dilute  alcohol. 
It  thus  will  not  be  precipitated  from  aqueous  solution  by  the  ad- 
dition of  alcohol.  This  is  a  point  of  difference  from  dextrin, 
which  is  precipitated  by  alcohol. 

ii.  Feliling's  Test. — Fehling's  reagent  is  made  up  in  two  parts. 
A  and  B,  which  are  mixed  in  equal  amounts  immediately  before 

217 


218  PHYSIOLOGICAL    CHEMISTRY 

using.  A  contains  copper  sulphate;  B  contains  sodium  hydrate 
and  sodium  potassium  tartrate  (Rochelle  salt).  The  solutions 
are  kept  separate,  as  after  mixing,  a  reduction  will  take  place  in 
the  course  of  time,  due  to  the  action  of  the  tartrate. 

Mix  equal  portions  (about  5  c.c.)  of  A  and  B.  A  temporary- 
whitish  precipitate  of  cupric  hydroxide  forms,  but  dissolves 
when  the  liquids  are  well  mixed,  the  solution  becoming  deep  blue. 
The  cupric  hydrate  forms  a  soluble  compound  with  the  tartrate 
present,  the  tartrate  thus  serving  to  hold  the  cupric  hydrate  in 
solution.  Heat  to  boiling.  No  change  occurs,  since  the  cupric 
hydrate  held  by  the  tartrate  does  not  decompose.  If  the  tartrate 
were  not  present,  the  cupric  hydrate  would  be  converted  into 
black  cupric  oxide  on  boiling.  To  the  hot  liquid  add  a  few  drops 
of  dilute  dextrose  solution  and  boil.  If  no  change  is  observed, 
add  more  dextrose  and  boil  again.  Eepeat  until  a  reaction  is 
obtained.  A  precipitate  forms  which  may  be  yellow  at  first 
(cuprous  hydroxide).  On  further  boiling  this  is  converted  into 
red  or  brownish  red  cuprous  oxide.  This  is  one  of  the  most 
widely  used  tests  for  reducing  sugars. 

iii.  Barfoed's  Test  is  similar  in  principle  to  Fehling's.  The 
solution  contains  copper  acetate  and  acetic  acid,  the  copper  being 
reduced  by  monosaccharides  as  in  the  Fehling  test.  A  disac- 
charide  usually  gives  the  Barfoed  reaction  only  on  prolonged 
boiling,  which  hydrolyzes  the  disaccharide  to  simple  sugars.  If 
properly  applied,  Barfoed's  test  may  be  used  to  distinguish  be- 
tween mono-  and  disaccharides  but  a  concentrated  maltose  solu- 
tion will  give  a  quicker  reduction  than  a  dilute  glucose  solution, 
hence  unless  experimental  conditions  are  carefully  controlled, 
erroneous  conclusions  may  result.  Place  about  5  c.c.  of  Bar- 
foed's solution  in  a  test  tube  and  heat  to  boiling.  Add  diluted 
dextrose  solution  (1  to  5)  a  few  drops  at  a  time,  heating  after 
each  addition.    A  red  precipitate  of  cuprous  oxide  forms. 

iv.  Haines'  Test  is  similar  to  Fehling's,  except  that  glycerine 
is  used  in  place  of  Rochelle  salt  and  potassium  hydroxide  in  place 
of  sodium  hydroxide. 


CARBOHYDRATES  219 

V.  Nylander's  Reagent  contains  bismuth  subnitrate,  Rochelle 
salt  and  potassium  hydroxide.  To  about  3  c.c  of  undiluted  dex- 
trose solution  add  about  I/2-I  c.c  of  Nylander's  reagent  and  boil 
3-4  minutes.  The  liquid  darkens  as  the  result  of  the  formation 
of  metallic  bismuth.  This  test  is  sometimes  called  the  Almen,  or 
Almen-Nylander  test. 

vi.  The  Phenylhydrazine  Test  depends  on  the  formation  of 
osazones,  relatively  insoluble  compounds  of  phenylhydrazine  and 
sugars. 

In  a  test  tube  mix  5  drops  phenylhydrazine,  10  drops  glacial 
acetic  acid  and  15  drops  saturated  sodium  chloride.  To  the  re- 
sulting solid  add  about  3  c.c.  of  dextrose  solution  and  boil  for 
2-3  minutes.  Allow  to  stand.  Examine  crystals  under  the  micro- 
scope and  draw.  The  osazones  form  yellow  needles  which  often 
group  together  in  rosettes,  sheaves,  or  fans.  Many  of  the  sugars 
may  be  identified  by  the  crystal  form  or  the  melting  point  of 
their  osazones. 

vii.  Moliscli's  Test. — To  about  5  c.c.  of  dextrose  solution  add  2 
drops  of  Molisch's  reagent  (15%  alcoholic  cc  naphtliol).  In- 
cline the  test  tube  and  pour  concentrated  sulphuric  acid  care- 
fully down  the  side  to  form  a  layer  at  the  bottom  of  .the  tube. 
Notice  the  reddish  violet  ring  at  the  junction  of  the  two  liquids. 
This  test  is  extremely  delicate,  but  is  given  by  various  substances 
other  than  carbohydrates.  A  negative  result  is  good  evidence 
that  carbohydrates  are  absent.  A  positive  test,  however,  may 
be  due  to  other  substances  or  to  shreds  of  filter  paper  (cellulose). 

viii.  Heat  a  small  portion  of  dry  dextrose  in  a  test  tube.  Note 
the  brown  color  and  the  odor  of  caramel.  If  a  small  amount  of 
sugar  is  heated  in  a  test  tube  with  concentrated  potassium 
hydroxide,  a  similar  result  is  observed, — the  color  and  odor 
of  caramel  appearing.    This  is  known  as  Moore's  test. 

ix.  Optical  Activity. — Carefully  read  the  discussion  of  optical 
activity  in  the  text.  The  specific  rotation  of  dextrose  is  -|-52.5° 
at  20°  C.  provided  the  concentration  is  not  above  15%.  With  a 
polariscope  determine  the  rotary  power  of  the  dextrose  solu- 


220  PHYSIOLOGICAL   CHEMISTRY 

tion  furnished,  and  calculate  the  strength  of  the  solution  by 
means  of  the  following  formula: 

cc     .    100 


[oc]20°.L 

c  =  grams  per  100  cc. 

oc  =  observed  rotation 

20° 
[  oc  ]  '^jr    =  specific  rotation 

L  =  length  of  observation  tube  in  decimetres 

Record  the  observed  rotation.    By  means  of  the  formula  calcu- 
late the  weight  of  dextrose  per  100  cc. 

If  the  weight  of  an  unknown  sugar  in  100  cc  of  solution  is 
known,  the  specific  rotation  may  be  calculated  by  observing  the 
rotation,  and  substituting  in  the  following  formula : 

r     -,  20°  oc        .     100 

■^     ^    D  L    .       c 

By  referring  to  a  table  of  specific  rotations  the  sugar  under 
investigation  can  thus  be  identified. 

X.  Fermentation. — The  fermentation  test  is  useful  in  detect- 
ing the  presence  of  many  sugars.  If  dry  yeast  is  used,  the  yeast 
solution  must  be  "set"  the  day  before  using  and  kept  warm  at 
least  over  night.  Compressed  yeast  may  be  used  immediately. 
Gently  rub  up  the  yeast  with  the  sugar  solution  to  be  tested, 
making  a  homogeneous  mixture. 

Fill  an  Einhorn  fermentation  tube  with  this  mixture.  Make 
sure  that  all  air  is  removed  from  the  tube.  When  the  tube  is 
full,  pour  out  most  of  the  liquid  from  the  bulb.  Otherwise  it  will 
overflow  into  the  incubator  in  case  fermentation  takes  place. 
Label  the  tube  with  your  name  and  the  kind  of  sugar  under  ex- 
amination and  place  in  an  incubator  at  38°-40°  C.  until  the  next 
class  period.  Remove  the  tube  and  add  3-4  cc  cone  sodium 
hydrate  from  a  pipette  in  such  a  way  that  it  will  enter  the  upright 
portion  of  the  tube.    Be  careful  that  the  gas  in  the  tube  does 


CARB0HYD1?ATES  221 

not  escape  and  that  no  air  is  admitted.     The  alkali  will  absorb 
the  CO2,  and  the  liquid  will  rise  in  the  tube. 

The  presence  of  alcohol  in  the  fermented  liquid  may  be  shown 
by  neutralizing  and  distilling.  This  is  most  conveniently  done 
with  the  combined  material  of  the  whole  class,  the  distillation 
being  performed  by  the  laboratory  attendant.  To  about  10  c.c  of 
the  distilled  liquid  add  5-6  drops  of  10%  sodium  hydrate  (no 
more).  Warm  to  about  50°  C.  (This  point  may  be  determined 
by  feeling  the  test  tube.  Fifty  degrees  feels  hot,  but  still  can 
be  borne  by  the  hand).  Add  iodine  solution  drop  by  drop  until 
the  liquid  has  a  faint  brown  tinge.  Allow  the  tube  to  stand. 
Notice  odor  of  iodoform.     This  is  a  test  for  alcohol. 

b.  Levulose. —  (Fructose.) 

Repeat  the  following  tests  made  on  dextrose,  using  levulose 
solution, 

i.  Solubility. — The  solubilities  of  levulose  are  similar  to  those 
of  dextrose.    The  student  need  not  repeat  the  tests. 

ii.  Fehling's  Test. 

iii.  Phenylhydrazine  Test. — Dextrose  and  levulose  form  the 
same  osazone,  so  that  they,  cannot  be  distinguished  one  from  the 
other  by  this  test. 

iv.  Molisch  Test. 

V,  Optical  Activity.- — The  specific  rotation  of  levulose  is — 93°. 
Determine  the  amount  of  levulose  in  a  solution  furnished. 

vi,  F ermentation. — ^Levulose  ferments  readily, 

c.  Galactose. — 

This  sugar  gives  the  usual  reduction  tests  and  forms  an  osa- 
zone. It  may  be  distinguished  from  dextrose  and  levulose  by 
the  mucic  acid  test.  Lactose  also  gives  this  test,  as  galactose 
makes  up  one-half  of  the  lactose  molecule.  To  distinguish  be- 
tween lactose  and  galactose  one  may  use  the  Barfoed  test,  which 
reacts  with  lactose  only  after  prolonged  boiling. 

i.  Mucic  Acid  Test. — To  50  c.c.  of  galactose  solution  add  10 
c.c.  concentrated  nitric  acid  and  evaporate  on  the  water  bath  to 


222  PHYSIOLOGICAL    CHEMISTRY 

about  3  c.c.  or  less.  The  fluid  should  be  clear  at  this  point,  and 
a  fine  white  precipitate  of  mucic  acid  should  form. 

II.  Pentoses.— Only  two  pentoses  are  of  physiological  in- 
terest, arabinose  and  xylose.  These  pentoses  give  the  reduction 
tests  and  form  osazones.  They  may  be  distinguished  from  dex- 
trose and  levulose  by  two  color  reactions,  the  orcin  and  phlo- 
roglucin  tests. 

a.  Arabinose. — The  arabinose  may  be  obtained  by  hydrolyzing 
gum  arable  by  boiling  for  several  hours  with  1-2%  sulphuric 
acid. 

i.  Orcin  Test. — Mix  equal  volumes  (about  2  c.c.)  of  arabinose 
solution  and  concentrated  hydrochloric  acid,  add  a  few  grains 
of  orcin  and  heat  on  the  water  bath.  In  the  presence  of  a  pen- 
tose, galactose,  or  glucuronic  acid  a  red  color  develops  which 
gradually  passes  through  reddish  blue  to  green.  The  color  alone 
is  not  sufficient  proof  of  the  presence  of  a  pentose.  To  confirm 
the  test  it  is  necessary  to  extract  the  liquid  with  amyl  alcohol; 
if  pentoses  are  present,  this  extract  will  contain  the  colored 
compound  which  should  show  an  absorption  band  between  the 
Fraunhofer  lines  C  and  D. 

ii.  TJie  pJdoroglucin  test,  commonly  called  Tollen's  reaction, 
is  similar  to  the  above  test  except  that  phloroglucin  is  used  in 
place  of  orcin.  The  absorption  band  of  the  amyl  alcohol  extract 
is  between  D  and  E. 

iii.  Final  proof  of  the  presence  of  a  particular  pentose  is  ob- 
tained by  determining  the  melting  point  of  its  osazone. 

II.  Disaccharides 

a.  Saccharose  (Sucrose). — Study  the  properties  of  saccharose 
as  follows : 

i.  Solubility.— {'^QQ  note  on  solubility  determinations  under 
Dextrose) .  Test  the  solubility  of  cane  sugar  in  water,  cold  alco- 
hol, hot  alcohol  (do  not  warm  over  a  flame, — dip  the  test  tube 
in  hot  water)  and  ether. 


CARBOHYDRATES  223 

ii.  Try  Feliling's  Test. — Saccharose  does  not  reduce  Fehling's 
solution  on  short  boiling  since  it  has  no  free  aldehyde  or  ketone 
group.  Try  boiling  for  several  minutes.  On  prolonged  boiling 
some  reduction  takes  place. 

iii.  Barfoed's  Test. — If  saccharose  does  not  reduce  Barfoed's 
solution  on  boiling  for  a  short  time,  try  the  effect  of  prolonged 
boiling.  Note  the  length  of  time  required  for  any  reaction  to 
take  place,  and  compare  it  with  the  time  in  which  dextrose 
brought  about  a  reduction. 

iv.  Nylander's  Test. 

V.  PJienylJiydrazine. — Cane  sugar  forms  no  osazone. 

vi.  Inversion  of  Saccharose. — By  boiling  with  dilute  acids  sac- 
charose may  be  broken  up  into  dextrose  and  levulose.  After 
ascertaining  that  the  saccharose  solution  will  not  reduce  Feh- 
ling's, take  another  sample  of  saccharose  solution,  acidify  slightly 
by  adding  about  1  c.c.  of  dilute  sulphuric  acid  and  heat  on  the 
water  bath  for  15  minutes.  Test  again  with  Fehling's  solution. 
The  saccharose  will  have  been  hydrolyzed  into  dextrose  and 
levulose,  and  the  liquid  should  reduce  Fehling's  solution. 

vii.  Optical  Activity  Before  and  After  Inversion. — Determine 
the  rotation  of  the  solution  of  saccharose.  Determine  the  rota- 
tion of  a  portion  of  the  same  solution  which  has  been  inverted. 
The  rotation  should  now  be  to  the  left,^  since  the  solution  contains 
equal  amounts  of  dextrose  and  levulose,  and  the  latter  is  stronger 
levorotatory  than  the  former  is  dextrorotatory, 

viii.  Perform  a  Fermentation  Test  with  Saccharose. — It  fer- 
ments readily. 

b.  Maltose. — Repeat  the  following  tests  performed  on  sac- 
charose, using  a  maltose  solution : 

i.  Solubility  in  ivater,  alcohol  and  ether. 
ii.  Fehling's. 
iii.  Barfoed's. 
iv.  Nylander's. 
V.  Phenylhydrazine. 
vi.  Fermentation. 


224  PHYSIOLOGICAL    CHEMISTRY 

c.  Lactose. — Perform  the  following  tests: 

i.  Solubility  in  water,  alcoJiol  and  etlier. 
ii.  FeJiling's. 

iii.  Barfoed's. 

iv.  Nylander's. 
V.  PJienyniydrazine. 

vi.  Fermentation. 

Lactose  does  not  ferment  with  ordinary  yeast.  This  test  is  of 
importance  in  identifying  lactose.  In  testing  for  lactose  a  con- 
trol should  be  run  with  a  lactose  solution,  to  make  sure  that  no 
lactose  splitting  enzyme  is  present  in  the  yeast  used. 

vii.  Lactose  may  also  be  distinguished  from  all  other  reducing 
sugars  except  galactose  by  the  mueic  acid  test.  Lactose  may  be 
distinguished  from  galactose  by  the  fermentation  test. 

III.  Polysaccharides 
1.  Starches. — 

a.  Potato  Starch. — Pare  a  small  potato  and  with  a  pocket 
knife  scrape  it  to  a  fine  pulp.  Mix  with  300-400  c.c.  of  distilled 
water  and  whip  up  thoroughly.  Strain  through  a  piece  of  cheese 
cloth  to  remove  the  coarse  particles.  The  starch  granules  will 
settle  rapidly  to  the  bottom  of  the  beaker.  Wash  once  or  twice 
by  decantation  and  examine  under  a  microscope.  Draw  the  gran- 
ules. Filter  off  a  portion  and  allow  it  to  dry  in  the  air.  Make 
the  following  tests  on  starch. 

i.  Solubility. — Refer  to  directions  for  determining  solubility  as 
given  under  Dextrose.  In  the  case  of  starch,  the  substance  is 
tested  for  in  the  filtrate  by  chemical  means.  Test  the  solubility 
of  starch  in  cold  water,  hot  water,  alcohol,  and  ether.  If  un- 
certain of  the  result,  filter  and  test  the  filtrate  for  starch  by  the 
iodine  test  as  described  below. 

ii.  Preparation  of  Starch  Solution. — Heat  about  75  c.c.  of  dis- 
tilled water  to  boiling.  To  this  add  about  1/2  gram  of  starch 
which  has  been  rubbed  to  a  thin  paste  with  a  small  amount  of 
cold  water.     Boil  very  slowly  for  about  15  minutes,  replacing 


CARBOHYDRATES  225 

water  lost  by  evaporation.  Use  the  resulting  opalescent  solution 
for  the  following  tests. 

iii.  Iodine  Test. — To  about  half  a  test  tube  of  distilled  water 
add  3-4  drops  of  iodine  solution.  Pour  3-4  drops  of  this  diluted 
solution  into  4-5  c.c.  of  the  starch  solution  prepared  in  ii.  Notice 
the  dark  blue  color.  This  is  due  to  a  starch-iodine  compound 
formed.  Iodine  gives  no  color  with  mono-  or  disaccharides.  To 
a  starch  solution  colored  blue  by  iodine  add  2-3  drops  of  dilute 
sodium  hydroxide.     The  color  is  destroyed. 

To  another  portion  of  the  blue  liquid  add  alcohol.  The  blue 
color  disappears. 

iv.  Perform  FeJiling's  Test. — Starch  does  not  reduce  Fehling's 
solution. 

v.  To  starch  solution  add  2-3  c.c.  tannic  acid  solution.  The 
starch  is  precipitated.  The  tannic  acid  solution  should  be  fresh, 
as  tannic  acid  solutions  decompose  on  standing. 

vi.  Starch  will  not  dialyze  through  a  parchment  membrane. 

vii.  Hydrolysis  of  Starch. — Place  about  25  c.c.  starch  solu- 
tion in  an  evaporating  dish,  add  10  drops  of  concentrated  hydro- 
chloric acid  and  boil  gently.  At  intervals  of  about  a  minute  re- 
move a  drop  or  tw^o  of  the  liquid  with  a  glass  rod  and  test  with  a 
drop  of  iodine  solution.  The  starch  is  broken  down  into  dex- 
trins  and  finally  into  simpler  carbohydrates  (maltoses  and  glu- 
cose). The  iodine  test  changes  from  blue  to  red,  and  finally  to 
no  color  as  the  hydrolj^sis  proceeds.  "When  iodine  no  longer  gives 
a  color,  make  the  hydrolyzed  starch  solution  alkaline  with  sodium 
hydrate  and  perform  a  Fehling's  test.  Reduction  indicates  the 
maltose  or  glucose  stage. 

2.  Dextrines.— 

i.  Test  the  solubility  of  dextrin  in  cold  water,  hot  water,  alco- 
hol and  ether. 

ii.  Iodine  Test. — Test  a  solution  of  dextrin  with  a  few  drops 
of  diluted  iodine  solution,  as  under  "starch."  Refer  to  the  re- 
sults obtained  on  the  hydrolysis  of  starch.  Try  the  effect  of 
heat;  of  alkali  and  of  alcohol  on  the  color  produced  with  iodine. 

iii.  Make  a  F elding' s  Test    on    dextrin   solution. — Remember 


226  PHYSIOLOGICAL    CHEMISTRY 

that  if  reduction  is  observed  it  may  be  due  to  the  presence  of 
maltose  or  dextrose  as  impurities  in  the  dextrin.  In  the  manu- 
facture of  dextrin,  small  amounts  of  maltose  and  dextrose  are 
likely  to  be  formed  by  complete  hydrolysis  of  a  portion  of  the 
starch.  Pure  dextrin  is  believed  not  to  reduce  Fehling's  solu- 
tion. 

iv.  To  dextrin  solution  add  alcohol.  Recall  the  effect  of  alco- 
hol on  dextrose. 

3.  Glycogen. — 

This  polysaccharide  may  be  obtained  from  the  liver  taken  from 
an  animal  immediately  after  death,  or  from  fresh  oysters.  The 
material  must  be  fresh,  as  otherwise  the  glycogen  will  have  been 
broken  down  to  glucose  by  the  tissue  enzymes.  The  liver  (or 
oysters)  is  thrown  into  boiling  water  slightly  acidulated  with 
acetic  acid.  After  boiling  a  few  minutes,  the  pieces  are  re- 
moved, ground  in  a  mortar  with  sand,  and  returned  to  the 
water  and  boiled  for  several  minutes.  The  glycogen  solution  is 
then  filtered  while  hot  from  the  coagulated  protein  material. 

i.  Test  glycogen  solution  with  iodine.  A  red  or  brown  color 
develops.  "Warm  gently  by  holding  the  test  tube  in  a  beaker  of 
water  heated  to  about  50°  C.  Remove  as  soon  as  the  color  disap- 
pears and  cool  under  the  tap.    The  color  should  reappear. 

ii.  Test  glycogen  for  reducing  sugar  with  Fehling's  solution. 
Glycogen  should  not  reduce,  but  a  solution  prepared  as  above 
often  will  do  so,  as  the  result  of  partial  hydrolysis  of  the 
material. 

iii.  To  10  c.c.  of  glycogen  solution  add  about  10  drops  of  con- 
centrated hydrochloric  acid  and  boil  for  about  10  minutes. 
Neutralize  carefully  with  sodium  hydrate  and  repeat  the  Fehling 
test.  The  glycogen  is  hydrolyzed  by  the  acid,  and  the  solution 
should  give  a  good  reduction. 


CHAPTER  IV 
FATS  AND  PHOSPHATIDES 

I.  Fats 

The  fats  found  in  the  animal  body  are  mainly  the  triglycerides 
of  palmitic,  stearic  and  oleic  acids.  Olive  oil  and  lard  are  con- 
venient materials  for  laboratory  study.  Butter  or  mutton  tallow 
may  be  used  with  equal  advantage. 

i.  Soluhility. — Test  the  solubility  of  olive  oil  and  lard  in  water, 
cold  and  hot  alcohol,  ether  and  chloroform.  To  3  c.e.  of  solvent 
add  2  drops  of  oil  or  a  small  piece  of  lard.  Note :  Do  not  heat 
alcohol  over  the  free  flame.  Heat  some  water  to  the  boiling  point 
in  a  small  beaker.  Turn  out  the  flame  and  warm  the  alcohol  by 
dipping  the  test  tube  in  the  hot  water. 

11.  Formation  of  Crystals. — Many  fats  will  crystallize  in  small 
needles  from  warm  alcohol,  benzol,  etc. 

iii.  Place  a  drop  of  oil  or  a  small  piece  of  lard  on  a  filter 
paper.  Observe  the  transparent  spot.  This  test  may  be  used  to 
detect  the  presence  of  a  fat  in  a  solvent.  On  evaporation  of  the 
solvent  a  transparent  spot  remains  on  the  paper  if  fat  was  pres- 
ent in  solution. 

iv.  Preparation  of  a  Neutral  Oil  (MatJiews). — Most  commer- 
cial fats  and  oils  contain  some  free  fatty  acid.  To  get  a  neutral 
oil  this  free  acid  must  be  neutralized. 

To  about  25  c.c.  of  95%  alcohol  in  a  small  flask  add  about  i/^ 
c.c.  phenolphthalein  solution.  Heat  to  boiling  on  the  steam  bath 
and  from  a  pipette  add  0.5%  NaOH  (dilute  10%  twenty  times) 
until  a  faint  pink  coloration  remains.  Now  add  6  c.c.  of  oil. 
Again  heat  to  boiling,  then  stopper  the  flask  and  shake  gently. 
The  alkaline  alcohol  becomes  colorless  as  the  alkali  is  neutralized 
by  the  fatty  acids  present.     Continue  the  addition  of  NaOH  as 

227 


228  PHYSIOLOGICAL    CHEMISTRY 

above,  shaking  after  each  addition  until  after  a  vigorous  shaking 
the  pink  color  in  the  alcohol  layer  just  remains.  Allow  the  two 
layers  to  separate  in  a  large  test  tube.  Draw  off  by  means  of  a 
pipette  the  layer  of  neutral  oil,  wash  it  by  shaking  gently  with 
one  or  two  portions  of  distilled  water  in  a  test  tube,  and  sav^  it 
for  use  in  the  experiments  following  where  neutral  oil  is  called 
for. 

V.  Emulsification. — The  property  of  forming  an  emulsion  is 
of  great  importance.  The  fat  is  held  in  suspension  in  the  liquid 
in  the  form  of  minute  droplets.  Study  the  conditions  affecting 
emulsification  as  follows: 

(a)  Shake  together  thoroughly  about  1  c.c.  of  neutral  oil  and 
a  few  c.c.  of  water.  The  oil  and  water  quickly  separate  into  two 
layers. 

(b)  Repeat  (a),  first  adding  a  small  amount  of  sodium  car- 
bonate solution  to  the  tube.  The  milky  appearance  of  the  water 
layer  indicates  that  a  portion  of  the  oil  has  emulsified,  but  the 
action  is  not  extensive.  To  neutral  oil  and  water  add  soap  solu- 
tion and  shake.  A  good  emulsion  forms,  indicating  the  favorable 
action  of  soap  on  emulsion  formation.  Upon  this  property  of 
soap  depends  in  large  measure  its  usefulness  in  cleansing.  Greasy 
material  on  the  surface  washed  is  emulsified,  and  the  insoluble 
"dirt"  thus  liberated  and  carried  away  by  the  water  and  the 
''suds." 

If  rancid  oil  (oil  which  contains  free  fatty  acid),  water  and  an 
alkali,  e.g.  Na^COg  are  shaken  together  a  good  emulsion  is  formed, 
since  the  mixture  contains  the  soap  produced  by  the  action  of  the 
fatty  acids  and  the  alkali. 

(c)  To  neutral  oil  and  water  add  albumin  solution  and  shake 
thoroughly.     Albumin  is  a  good  emulsifying  agent. 

(d)  To  neutral  oil  and  water  add  gum  arabic  (acacia)  solu- 
tion and  shake.    Gum  arabic  also  is  a  good  emulsifier. 

(e)  Lymph  has  the  power  of  aiding  in  the  formation  of  an 
emulsion.  This  may  be  demonstrated  (Ranvier's  experiment) 
by  bringing  drops  of  oil  and  lymph  together  under  the  micro- 


FATS   AND   PHOSPHATIDES  229 

scope.    Intense  activity  will  be  observed  at  the  juncture  of  the 
liquids,  and  the  oil  passes  into  an  emulsified  state. 

(f)  Examine  an  emulsion  under  the  microscope.  Observe  the 
tiny  globules  of  fat.  Examine  a  drop  of  milk  under  the  micro- 
scope. The  fat  in  milk  is  emulsified.  On  standing,  a  portion  of 
it  will  rise  to  the  surface.  This  layer  containing  much  fat  is 
known  as  cream. 

vi.  Sapo7iification  of  Fats. — If  a  fat  is  heated  with  an  alkali 
it  is  split  into  fatty  acids  and  glycerine.  The  liberated  fatty 
acids  unite  Avith  any  excess  of  alkali  to  form  soaps,  hence  the 
term  saponification. 

In  a  flask  heat  to  boiling  on  the  steam  bath  50  c.c.  of  alcoholic 
sodium  hydrate  and  add  5  c.c.  of  olive  or  cottonseed  oil  and  con- 
tinue heating.  The  saponification  is  complete  when  all  the  oil 
has  disappeared.  This  may  occur  almost  immediately.  When 
this  point  has  been  reached,  transfer  to  an  evaporating  dish,  add 
about  75  c.c.  of  water  and  heat  on  the  water  bath  to  drive  off 
the  alcohol.  When  the  alcohol  has  been  removed  (decide  this  by 
the  odor)  divide  the  liquid  into  three  parts.  Filter  if  necessary. 
To  one  portion  add  dilute  sulphuric  acid  and  warm  on  the  water 
bath.  The  acid  converts  the  soaps  into  free  fatty  acids  which 
form  an  oily  layer  at  the  surface  (oleic  acid).  If  a  palmitic  or 
stearic  acid  fat  is  used,  the  fatty  acids  separate  in  solid  form. 

To  a  second  portion  of  the  sodium  soap  solution  add  an  equal 
volume  of  saturated  sodium  chloride.  The  soap  is  "salted  out" 
since  it  is  insoluble  in  sodium  chloride  solution.  In  case  no  pre- 
cipitate forms  evaporate  the  solution  on  the  water  bath  to  a 
smaller  volume. 

To  the  third  portion  of  the  sodium  soap  solution  add  calcium 
chloride  solution.  The  precipitate  is  calcium  soap,  which  is 
more  insoluble  than  sodium  soap.  Hard  water,  which  contains 
calcium  salts,  is  unsuitable  for  washing  purposes,  for  the  cal- 
cium precipitates  soap  in  white  flakes,  and  thus  removes  it 
from  solution. 

vii.  Acrolein  Test. — If  a  fat  is  heated  with  boric  acid  or  potas- 
sium bisulphate,  acrolein  is  formed,  which  may  be  recognized  by 


230  PHYSIOLOGICAL    CHEMISTRY 

its  sharp,  disagreeable  odor.  Heat  a  small  amount  of  lard  or 
olive  oil  ynth  boric  acid  or  potassium  bisulphate  in  a  dry  test 
tube.  Continue  the  heating  until  the  material  has  become  prac- 
tically dry  and  note  the  penetrating  odor  of  acrolein.  Repeat 
the  test  using  a  few  drops  of  glycerine  in  place  of  the  lard.  The 
characteristic  acrolein  odor  again  will  be  observed.  Fats  give  the 
test  because  of  the  glycerine  which  they  contain.  Fatty  acids 
and  pure  soaps  do  not  give  the  test,  a  result  which  might  be 
expected  from  the  fact  that  they  contain  no  glycerine. 

II.  Phosphatides 

The  phosphatides  contain  glycerine,  phosphoric  acid,  choline 
and  fatty  acid  radicles.  Chemically  they  are  closely  allied  to  the 
fats  (see  lecture  notes) .  A  lecithin  will  be  studied  as  an  example 
of  this  group. 

Lecithins  occur  probably  in  all  animal  cells  and  are  especially 
abundant  in  the  brain,  which  contains  also  other  members  of  the 
group  of  phosphatids  and  from  which  these  substances  may  be 
prepared.  For  laboratory  purposes,  lecithin  may  be  obtained 
from  egg  yolk. 

a.  Lecithin. — 

i.  Preparation. — The  yolk  of  one  egg  is  allowed  to  stand  with 
30  c.c.  ether  over  night.  To  30  c.c.  of  ether  extract  of  egg  yolk  add 
50  c.c.  of  alcohol.  If  a  precipitate  forms  (another  extractive  sub- 
stance if  present  in  large  amounts  may  be  thrown  down  by  alco- 
hol) filter.  Evaporate  on  the  water  bath,  dissolve  the  residue  in 
15  c.c.  ether  and  filter.  Add  40  c.c.  acetone  to  the  filtrate.  The 
lecithin  is  precipitated.  Filter  it  off.  The  filtrate  contains  chol- 
esterol.   Use  the  lecithin  for  the  following  tests : 

ii.  Add  a  small  amount  to  1  c.c.  of  water.  Note  opalescence. 
The  mixture  may  be  filtered  unchanged,  although  the  lecithin 
does  not  form  a  true  solution. 

iii.  On  a  small  portion  make  the  acrolein  test.  Since  lecithin 
contains  glycerol,  a  positive  test  should  result. 

iv.  Fuse  about  %  of  the  lecithin  with  fusion  mixture,  dissolve 


FATS   AND   PHOSPHATIDES  231 

ill  dilute  nitric  acid  and  test  for  phosphate  with  ammonium 
molybdate.    A  positive  test  should  result. 

V.  Saponify  H  of  the  lecithin  by  heating  in  a  flask  on  the 
water  bath  with  20  c.c.  of  alcoholic  NaOH  for  about  15  minutes, 
replacing  the  alcohol  if  necessary.  Evaporate  to  dryness  and  dis- 
solve the  soap  in  water.  Boil  and  observe  the  soap  bubbles. 
Acidify  with  hydrochloric  acid  and  allow  to  stand,  warming  if 
necessary.  The  liberated  fatty  acids  precipitate  and  float  at  the 
surface. 

vi.  The  choline  portion  of  the  molecule  also  may  be  recognized 
by  appropriate  tests. 


CHAPTER  V 

PROTEINS 

All  poteins  contain  carbon,  hydrogen,  oxygen  and  nitrogen; 
some  contain  also  sulphur,  phosphorus,  iron  or  other  elements. 
These  elements  may  be  detected  by  the  methods  described  in  the 
chapter  on  the  elements. 

General  Protein  Reactions 

The  general  tests  for  the  detection  or  isolation  of  the  proteins 
are  divided  into  two  groups,  color  reactions  and  precipitation 
reactions. 

1.  Color  Reactions. — 

i.  Biuret  Test. — Prepare  two  test  tubes  each  containing  a  few 
cubic  centimeters  of  water.  To  one  test  tube  add  a  few  drops  of 
egg  albumin  solution.  To  each  tube  add  1-2  e.c.  saturated 
sodium  hydrate  and  a  few  drops  of  copper  sulphate  solution 
which  has  been  diluted  until  it  has  only  a  faint  blue  color.  Such 
a  copper  sulphate  solution  can  be  prepared  by  adding  a  few 
drops  of  copper  sulphate  solution  to  a  half  test  tube  of  distilled 
water.  Notice  the  lavender  or  violet  color  in  the  albumin  tube, 
and  compare  it  with  the  color  of  the  control.  This  reaction  is 
very  delicate.  It  also  may  be  performed  by  adding  the  alkali 
to  the  protein  solution,  inclining  the  test  tube  slightly  and  allow- 
ing the  copper  sulphate  solution  to  flow  down  the  side  of  the 
tube  so  as  to  form  a  layer  on  the  surface  of  the  liquid.  A  lav- 
ender ring  forms  at  the  juncture  of  the  two  liquids.  The  biuret 
test  is  given  by  any  substance,  protein  or  otherwise,  which  con- 
tains two  CONII2  groups  joined  either  directly  or  by  a  single 
carbon  or  nitrogen  atom,  and  also  by  some  other  similar  group- 
ings.   The  test  is  named  from  the  fact  that  it  is  given  by  biuret, 

232 


PROTEINS  233 

CONH,  .  NH  .  CONHo,  a  substance  obtained  by  heating  urea  to 
180°  C. 

ii.  Millon's  Reaction.- — To  a  few  cubic  centimeters  of  albumin 
solution  add  2-3  (no  more)  drops  of  Millon's  reagent  (1  pt.  by 
weight  of  Hg.  dissolved  in  2  pts.  of  cone.  HNO3  and  diluted  with 
two  volumes  of  water).  A  yellowish  or  white  precipitate  forms. 
Heat  the  solution  carefully.  The  precipitate  will  turn  pink  or 
red.  Repeat  the  test,  first  adding  sodium  chloride  solution  to  the 
albumin  solution.  The  test  fails, — that  is,  the  precipitate  no 
longer  turns  pink.  This  fact  should  be  borne  in  mind  in  testing 
for  protein  in  a  liquid  containing  sodium  chloride.  Shake  up  a 
small  amount  of  dry  casein  with  w^ater  and  apply  the  Millon  test. 
The  test  is  given  by  solid  proteins  as  well  as  by  proteins  in  solu- 
tion. Perform  the  test  on  a  dilute  solution  of  phenol.  A  beau- 
tiful red  color  results.  The  reaction  is  given  by  substances  con- 
taining a  hydroxyphenyl  group  — OJI^  .  OH.  Most  proteins 
contain  tyrosine,  a  substance  possessing  this  grouping,  and  it  is 
because  of  the  presence  of  this  substance  that  the  proteins  give 
the  Millon  reaction. 

iii.  XantJioproteic  Eeadion. — To  a  few  cubic  centimeters  of 
egg  albumin  solution  add  concentrated  nitric  acid.  Warm  the 
mixture  until  the  whitish  precipitate  has  dissolved.  The  solu- 
tion is  yellow.  Cautiously  add  ammonia  until  the  reaction  is 
alkaline  and  observe  the  deepening  of  the  color  to  orange.  Re- 
peat the  test  using  a  small  portion  of  dry  casein.  Invert  the 
stoppered  bottle  of  concentrated  nitric  acid  slightly  so  as  to  get 
a  drop  on  the  glass  stopper.  Carefully  apply  the  stopper  mois- 
tened with  nitric  acid  to  a  small  area  on  the  palm  of  the  hand. 
In  a  moment  pour  on  the  yellow  spot  a  drop  or  so  of  ammonia. 
Observe  the  orange  spot.  Rinse  off  the  hand  carefully  under  the 
tap.  The  test  is  given  by  substances  containing  a  benzene  ring 
and  is  due  to  the  formation  of  certain  nitro-compounds.  Most 
proteins  contain  amino  acids  in  which  there  is  a  benzene  ring, 
and  thus  will  respond  to  this  test.  This  is  also  true  of  the  pro- 
teins of  the  skin. 

iv.  Adamkiewicz,  or  Hopkins-Cole  Reaction. — Mix  2-3  c.c.  of 


234  PHYSIOLOGICAL    CHEMISTRY 

albumin  solution  with  an  equal  volume  of  glyoxylic  acid  solution. 
Add  an  equal  volume  of  concentrated  sulphuric  acid,  pouring  it 
down  the  inside  of  the  test  tube  so  as  to  make  a  layer  beneath 
the  aqueous  solution.  A  reddish  violet  or  purple  ring  will 
develop  at  the  juncture  of  the  two  liquids.  Shake  the  tube  gently 
so  as  to  mix  the  two  layers.  The  color  will  spread  throughout 
the  entire  solution. 

This  test  is  often  performed  by  adding  glacial  acetic  acid  in 
place  of  a  solution  of  glyoxylic  acid,  as  glyoxylic  acid  is  usually 
present  in  glacial  acetic  acid  as  an  impurity.  It  is  more  satis- 
factory, however,  to  use  a  solution  of  glyoxylic  acid,  which  may 
be  prepared  by  reducing  oxalic  acid  with  sodium  amalgam  or 
magnesium.  The  reaction  is  due  to  tryptophane,  which  is  present 
in  most  proteins. 

V.  Molisch  Test. — The  student  is  already  familiar  with  the 
Molisch  test  from  his  work  on  carbohydrates.  It  is  given  by 
many  proteins  and  is  supposed  to  indicate  the  presence  of  a  car- 
bohydrate group  in  the  protein  molecule.  To  about  3-4  c.c.  of 
albumin  solution  add  2-3  drops  of  15%  alcoholic  oc  naphthol. 
Incline  the  tube  and  pour  down  the  side  a  few  cubic  centimeters 
of  concentrated  sulphuric  acid  to  form  a  layer  at  the  bottom 
of  the  tube.    Observe  the  red  ring. 

2.  Precipitation  Tests. 

i.  By  Heat. — Heat  about  3  c.c.  of  albumin  solution  to  boiling. 
If  no  precipitation  occurs,  add  1  drop  of  1%  acetic  acid  and  boil 
again,  repeating  the  process  until  coagulation  occurs.  Eepeat 
the  experiment,  adding  about  1  c.c.  saturated  sodium  chloride 
solution  to  the  albumin  solution.  The  presence  of  salts  is  fa- 
vorable for  coagulation. 

In  removing  the  protein  from  a  solution  the  acid  is  added  after 
heating,  as  otherwise  acid  metaprotein,  which  does  not  coagulate 
on  boiling,  may  be  formed.  Proteoses,  peptones,  casein  and  a  few 
other  proteins  are  not  coagulated  by  heat.  Heat  another  portion 
of  albumin  solution,  first  adding    1-2    drops    of    concentrated 


PROTEINS  235 

sodium  hydrate.  No  coagulation  occurs,  as  proteins  do  not  coag- 
ulate if  heated  in  alkaline  solution. 

ii.  Concentrated  Blineral  Acids. — Prepare  three  test  tubes  each 
containing  a  few  cubic  centimeters  of  albumin  solution.  Add 
concentrated  sulphuric,  hydrochloric,  and  nitric  acid  respectively 
drop  by  drop  to  the  three  tubes,  and  record  the  results  in  each. 
The  albumin  is  precipitated  in  each  tube.  The  coagulation  with 
nitric  acid  may  be  used  to  detect  extremely  small  amounts  of 
protein.  Dilute  1-2  c.c.  of  albumin  solution  with  several  volumes 
of  water.  To  a  few  cubic  centimeters  of  this  dilute  solution  in  a 
test  tube,  add  concentrated  nitric  acid  carefully  from  a  pipette. 
Caution. — In  taking  up  concentrated  acids  in  a  pipette,  great 
care  should  be  exercised  to  avoid  drawing  the  acid  into  the 
mouth.  Be  sure  the  point  of  the  pipette  is  kept  well  below  the 
surface  of  the  acid,  which  should  have  been  poured  into  a  clean 
test  tube  before  being  drawn  up  into  the  pipette.  Do  not  fill 
the  pipette  more  than  half  full  of  acid. 

Run  the  nitric  acid  slowly  into  the  albumin  solution  from  the 
pipette,  keeping  the  point  of  the  pipette  at  the  bottom  of  the 
test  tube.    This  facilitates,  the  formation  of  two  layers. 

Observe  the  cloudy  ring  at  the  juncture  of  the  two  liquids. 
Performed  in  this  way,  the  test  is  known  as  the  Heller  Ring  test, 
and  is  used  to  detect  the  presence  of  protein  in  urine. 

iii.  AlcoJiol. — To  a  few  cubic  centimeters  of  albumin  solution 
add  alcohol.    The  albumin  is  precipitated. 

iv.  Heavy  Metals. — To  small  portions  of  albumin  solution  add 
solutions  of  copper  sulphate,  mercuric  chloride  and  lead  acetate. 
The  albumin  is  precipitated  by  each  reagent.  Egg  white  is  used 
as  an  antidote  in  eases  of  poisoning  by  "blue  vitriol,"  "corrosive 
sublimate,"  etc.,  since  it  forms  insoluble  compounds  with  these 
metal  salts  which  then  can  be  pumped  from  the  patient's  stomach 
with  a  stomach  pump,  or  removed  by  vomiting.  The  salts  of  most 
heavy  metals  will  precipitate  proteins,  in  the  same  way  as  those 
tested  above. 

V.  Acetic  Acid  and  Potassium  Ferrocyanide. — To  a  few  cubic 
centimeters  of  albumin  solution  add  5-10  drops  of  10%  acetic 


236  PHYSIOLOGICAL    CHEMISTRY 

acid  and  then,  drop  by  drop,  potassium,  ferrocyanide.  A  pre- 
cipitate forms.  Avoid  an  excess  of  the  reagent,  as  the  precipitate 
may  be  redissolved.  This  test  is  very  delicate.  Zinc  also  will  give 
a  similar  precipitate  with  ferrocyanide,  a  fact  which  should  be 
borne  in  mind  if  the  liquid  to  be  tested  has  been  kept  in  a  zinc 
lined  can. 

vi.  Alkaloidal  Reagents.— Preipave  six  tubes  of  albumin  solu- 
tion and  test  the  precipitating  power  of  each  of  the  following 
reagents.  Add  a  few  drops  of  the  reagent  at  a  time,  until  a 
precipitate  forms,  avoiding  excess,  as  certain  of  the  precipitates 
are  soluble  in  excess  of  the  reagent :  picric  acid,  tannic  acid  and 
trichloracetic  acid.  Acidify  the  remaining  three  tubes  with  a  few 
drops  of  dilute  hydrochloric  acid  before  making  the  tests  with : 
phosphotungstic  acid,  phosphomolybdic  acid  and  potassium  mer- 
curic iodide.  An  approximate  estimation  of  the  albumin  in  a 
solution  may  be  made  by  precipitating  the  protein  in  a  specially 
graduated  tube,  called  an  Esbaeh  tube,  by  adding  Esbach's  re- 
agent, which  contains  picric  and  citric  acids. 

vii.  Ammonium  or  Magnesium  Sulphate,  Sodium  CMoride,  etc. 
"Salting  out." 

(a)  To  about  3  c.c.  of  blood  serum  in  a  test  tube  add  an  equal 
volume  of  water,  and  then  powdered  magnesium  sulphate,  (shak- 
ing) until  no  more  will  dissolve.  There  should  be  considerable 
excess  salt  in  the  tube  to  insure  saturation.  Globulins  are  thrown 
down.  Filter  off  the  liquid.  Test  the  precipitate  for  protein  by 
Millon's  reaction.  Test  a  portion  of  the  filtrate  by  the  xantho- 
proteic test,  not  forgetting  the  addition  of  ammonia.  A  positive 
test  should  result,  since  the  filtrate  still  contains  serum  albumin, 
which  is  not  precipitated  under  the  above  conditions.  To  the 
remainder  of  the  filtrate  add  1-2  drops  of  dilute  acetic  acid.  A 
precipitate  results,  as  albumins  are  precipitated  by  saturating 
with  magnesium  sulphate  in  acid  solution. 

(b)  Repeat  (a)  using  solid  ammonium  sulphate  in  the  place 
of  magnesium  sulphate.  A  copious  precipitate  should  form.  Fil- 
ter and  test  filtrate  and  precipitate  for  protein  as  above.  The 
precipitate  should  give  a    positive   protein    test.      The    filtrate 


PROTEINS  237 

should  give  a  negative  protein  test  as  saturating  with  ammonium 
sulphate  even  in  neutral  solution  precipitates  both  albumins  and 
globulins.  To  a  portion  of  the  filtrate  add  1-2  drops  of  acetic 
acid.  No  precipitate  should  form,  since  the  albumin  already  has 
been  precipitated. 

(c)  To  a  small  portion  of  blood  serum  add  an  equal  volume  of 
saturated  ammonium  sulphate  solution,  thus  producing  a  mix- 
ture half  saturated  with  this  s^lt ;  this  will  have  the  same  effect  as 
saturating  with  magnesium  sulphate. 

viii.  Pour  2  or  3  drops  of  blood  serum  into  a  large  beaker  of 
distilled  water.  The  cloudiness  is  due  to  the  precipitation  of  a 
protein  (globulin)  which  is  soluble  in  the  blood  serum  because  of 
the  presence  of  certain  salts.  On  dilution  the  protein  precipi- 
.  tates.  The  same  results  may  be  obtained  if  the  salts  are  removed 
by  dialysis. 

Individual  Groups — Simple  Proteins 

1.  Albumins. — The  solution  of  egg  white,  and  also  the  blood 
serum  used  for  the  general  protein  tests  contained  both  albumins 
and  globulins.  These  two  classes  of  proteins  may  be  separated 
from  one  another  by  saturating  with  magnesium  sulphate,  or  half 
saturating  with  ammonium  sulphate  in  neutral  solution.  Either 
method  causes  globulin  to  precipitate.  Saturating  with  mag- 
nesium sulphate  will  precipitate  both  groups  if  the  solution  is 
acid.  Saturating  with  ammonium  sulphate  throws  down  both 
groups  in  neutral  solution.  Some  of  the  plant  albumins  and 
globulins  are  exceptions  to  the  above  statements. 

i.  Preparation  of  Albumin  Crystals. — Beat  up  5  c.c.  of  white 
of  egg  to  break  the  reticulum.  Dilute  with  ten  volumes  of  water 
and  strain  through  gauze.  Add  an  equal  volume  of  saturated 
ammonium  sulphate.  Label  with  your  name  and  leave  in  the 
ice  box  until  the  next  laboratory  period.  Filter  off  the  precipi- 
tated globulin  and  to  the  filtrate  add  saturated  ammonium  sul- 
phate until  the  liquid  becomes  turbid.  Now  add  distilled  water 
in  very  small  portions  until  the  turbidity  has  just  disappeared. 
Add  drop  by  drop  10%  acetic  acid  saturated  with  ammonium 


238  PHYSIOLOGICAL    CHEMISTEY 

sulphate  until  a  precipitate  is  obtained.  Again  place  in  the  ice 
box  until  the  next  period.  On  standing,  the  precipitate,  which 
is  at  first  amorphous,  will  become  crystalline.  Examine  under 
the  microscope.  The  crystals  are  small,  and  look  much  like  sand 
grains.  If  similarly  prepared,  serum  albumin  and  lactalbumin 
crystals  may  be  obtained. 

ii.  Solubility  of  Egg  Albumin. — Test  the  solubility  of  pow- 
dered egg  albumin  in  water  and  10%  HCl.  Recall  notes  on  solu- 
bility determinations  under  dextrose.  A  filtrate  may  best  be 
tested  for  protein  by  one  or  more  of  the  color  reactions.  Recall 
that  albumins  are  precipitated  in  neutral  solution  by  saturating 
with  ammonium  sulphate,  but  not  with  magnesium  sulphate,  or 
sodium  chloride,  or  on  half  saturation  with  ammonium  sulphate. 
If  the  solution  is  acid,  however,  in  these  latter  cases  some  albumin 
is  precipitated.  Recall  also  that  albumin  is  coagulated  by  heat 
and  is  precipitated  by  various  reagents  such  as  alcohol,  mineral 
acids,  etc. 

iii.  Recall  the  color  reactions  of  the  albumins  as  determined 
under  general  protein  tests. 

iv.  Coagulation  Temperature  of  Egg  Albumin  Solution. — Fill 
a  large  beaker  half  full  of  tap  water.  In  this  beaker  place  a 
smaller  beaker  or  an  Erlemeyer  also  about  half  full  of  water.  If 
the  inner  vessel  is  not  supported  by  fitting  into  the  large  beaker, 
arrange  the  amount  of  water  in  this  inner  vessel  so  that  it  will 
not  sink  to  the  bottom  of  the  large  beaker.  Place  a  test  tube 
c<  ntaining  about  five  cubic  centimeters  of  clear,  fresh  albumin 
solution  in  the  inner  vessel,  and  add  to  the  albumin  solution 
about  1  c.c.  of  saturated  sodium  chloride  and  a  few  drops  of  1% 
acetic  acid.  "While  your  partner  carefully  warms  the  water  in 
the  outer  beaker,  observe  the  thermometer  and  the  albumin  solu- 
tion.- The  point  at  which  it  becomes  cloudy  is  taken  as  the  coagu- 
lation temperature  of  the  protein  under  examination. 

2.  Globulins. — ^As  has  been  observed  above,  globulins,  at  least 
those  of  animal  origin,  are  precipitated  from  neutral  solution  by 
saturating  with  MgS04  or  half  saturating  with  (NH4)2S04. 

i.  Flant  Globulins. — Globulins  are  found  in  many  plants  and 


PROTEINS  239 

may  be  extracted  with  dilute  salt  solutions.  An  example  of  this 
class  is  Edestin,  which  may  be  prepared  by  extracting  hemp 
seed  with  5%  sodium  chloride  solution. 

ii.  Globulins  of  the  Blood  Plasma. — If  time  permits,  the  vari- 
ous blood  globulins  may  be  fractionally  precipitated  from  blood 
plasma.  Quarter  saturation  brings  down  fibrinogen,  half  satura- 
tion precipitates  practically  all  the  remaining  globulins  of  which 
there  may  be  several.  The  filtrate  from  the  globulins  still  con- 
tains serum  albumin,  which  may  be  precipitated  by  acidifying 
slightly,  or  by  saturating  with  ammonium  sulphate.  Fibrinogen 
also  may  be  precipitated  by  half  saturating  with  NaCl,  differing 
in  this  respect  from  the  other  globulins  which  require  full  satura- 
tion for  precipitation. 

Fibrin. — On  beating  freshly  drawn  blood  with  a  rod,  the 
fibrinogen  separates  as  shreds  of  fibrin  which  gather  on  the  rod, 
and  may  be  washed  free  of  corpuscles.  Examine  a  piece  of  fibrin. 
Note  its  elasticity.  It  possesses  the  general  properties  of  the 
globulins.  Test  its  solubility  in  water  and  10%  NaCl.  Filter  off 
the  liquid  and  test  it  for  protein  by  an  appropriate  test  to  ascer- 
tain whether  or  not  any  fibrin  has  dissolved.  In  choosing  your 
test,  recall  the  effect  of  sodium  chloride  on  certain  of  the  color 
tests. 

Like  other  globulins,  fibrin  is  soluble  in  dilute  salt  solutions, 
but  complete  solution  takes  place  only  after  several  days'  stand- 
ing. 

On  small  pieces  of  solid  fibrin  suspended  in  water  try  the 
Millon  and  the  xanthoproteic  tests. 

Myosin.— This  muscle  globulin  may  be  prepared  from  the  mus- 
cle of  a  freshly  killed  rabbit,  by  grinding  in  a  mortar  with  5% 
MgS04,  preferably  after  washing  out  the  blood  vessels  with 
physiological  saline.  The  extract  contains  myosin  and  also  other 
proteins,  e.g.  paramyosinogen,  from  which  it  may  be  separated 
by  fractional  precipitation  with  MgS04.  At  50%  concentration, 
paramysinogen  precipitates. 

If  the  concentration  of  the  filtrate  is  increased  to  94%,  myosin 


240  PHYSIOLOGICAL    CHEMISTRY 

is  thrown  down.  It  may  be  prepared  and  its  reactions  studied 
if  time  permits. 

The  solid  meat  residue  left  after  extracting  the  myosin  is 
known  as  "muscle  stroma. "  It  contains  some  coagulated  myosin, 
and  probably  members  of  other  groups.  Test  it  by  the  xantho- 
proteic reaction  and  Millon's  test. 

iii.  NeuroglobuUns. — Two  and  perhaps  three  globulins  occur 
in  the  brain.  They  may  be  prepared  by  extraction  with  salt  solu- 
tions and  occur  chiefly  in  the  grey  matter  and  the  axis  cylinders. 

3.  Prolamines. — The  best  known  member  of  this  group  of 
alcohol  soluble  proteins  is  the  gliadin  of  wheat. 

Two  students  may  work  together.  To  about  50  gms.  of  flour 
add  in  small  portions  enough  water  to  make  a  dough.  Knead 
this  thoroughly  in  running  water  until  it  is  washed  free  of 
starch.  Press  out  as  much  water  as  possible  from  the  solid 
residue,  which  is  known  as  gluten.  Add  250  e.c.  of  70%  alcohol 
(prepare  this  by  diluting  185  c.c.  desk  alcohol,  95%,  to  250  e.c.) 
and  knead  up  thoroughly  with  the  hand.  Shake  often,  and  allow 
to  stand  until  the  next  laboratory  period,  or  extract  for  an  hour 
on  the  water  bath.  Filter  off  the  gliadin  solution  from  the  residue 
which  is  mainly  glutenin.  Evaporate  the  gliadin  solution  on  the 
water  bath.  On  the  residue  make  the  following  tests.  Test  its 
solubility  in  water  and  10%  NaCl.  Try  the  Millon  and  xantho- 
proteic tests. 

4.  Glutelins. — The  only  member  of  this  group  which  has 
been  carefully  studied  is  the  glutenin  of  wheat.  It  may  be  pre- 
pared from  wheat  flour  by  allowing  0.2%  KOH  to  act  on  the 
insoluble  residue  left  after  the  gliadin  has  been  completely  ex- 
tracted. This  alkaline  extract  is  then  neutralized  with  HCl, 
"when  the  glutenin  precipitates. 

5.  Albuminoids. — These  proteins  are  distinguished  by  their 
iiisolubility  in  neutral  solvents.  They  form  the  principal  or- 
ganic components  of  the  supporting  framework  of  the  body  and 
of  hair,  nails,  horn,  etc. 

i.  Keratin. —  (a)  Test  the  solubility  of  horn  in  water  and  10% 
NaCl. 


PROTEINS  241 

(b)  Place  a  few  pieces  of  horn  in  a  few  cubic  centimeters  each 
of  aitificial  gastric  and  pancreatic  juice,  add  a  small  amount  of 
toluol,  and  set  in  the  incubator  until  the  next  period.  Horn  is 
not  digested  by  gastric  or  pancreatic  juice.  Conclusions  as  to 
the  digestibility  of  horn  are  drawn  from  the  appearance  of  the 
pieces.  It  is  not  possible  to  use  the  color  tests  for  ascertaining 
whether  or  not  horn  has  been  digested,  as  the  pancreatic  and 
gastric  ferment  preparations  themselves  contain  soluble  proteins 
in  amounts  sufficient  to  give  positive  color  reactions. 

(c)  Suspend  small  pieces  of  horn  in  water  and  try  the  Millon 
and  xanthoproteic  tests.  Test  for  loosely  combined  sulphur.  All 
of  these  tests  should  be  positive. 

ii.  Collagen. — Collagen  may  be  prepared  from  the  tendon  of 
Achilles  of  an  ox  by  dissolving  out  the  mucoid  with  lime  water. 
After  several  days'  extraction,  the  residue  is  mainly  collagen. 
Collagen  is  insoluble,  only  slightly  digestible  and  gives  certain  of 
the  protein  color  tests.  On  boiling  for  some  time  it  is  converted 
into  gelatine. 

Properties  of  Gelatine. — With  gelatine  prepared  as  above  or 
with  commercial  gelatine  perform  the  following  experiments : 

Test  its  solubility  in  cold  water.  It  only  swells  up.  Heat. 
The  gelatine  dissolves.  On  cooling,  if  the  solution  is  concen- 
trated, it  solidifies  to  a  jelly. 

Gelatine  digests  in  gastric  and  pancreatic  juice. 

iii.  Elastin. — Elastin  may  be  prepared  from  the  ligamentum 
nuchae  of  the  ox.  The  ligament  is  cut  into  small  pieces  and 
washed  with  10%  NaCl  two  or  three  times,  and  then  with  running 
water  for  48  hours.  It  is  then  extracted  with  half  saturated  lime 
water  for  two  days.  The  material  then  is  boiled  for  at  least  two 
hours  with  dilute  acetic  acid  to  remove  collagen.  The  residue  is 
mainly  elastin.  It  is  insoluble,  somewhat  digestible,  exhibits  a 
remarkable  elasticity,  and  responds  to  several  of  the  protein 
color  tests. 

6.  Histones. — The  globin  Avhich  forms  a  part  of  the  hemo- 
globin molecule  is  considered  to  be  a  histone  by  most  authorities. 


242  PHYSIOLOGICAL    CHEMISTRY 

It  is  easily  split  off  from  hemoglobin  by  the  action  of  dilute  hydro- 
chloric acid. 

The  histones  give  the  biuret  test,  usually  only  a  faint  Millon 
reaction,  are  precipitated  from  neutral  solution  by  alkaloidal 
reagents,  and  form  precipitates  if  added  to  a  protein  solution. 
They  are  acted  on  by  the  digestive  juices.  They  contain  a  high 
percentage  of  diamino  acids. 

7.  Protamines. — The  protamines  have  been  found  only  in 
the  spermatozoa  of  fish.  The  protamines  give  the  biuret  test,  but 
most  of  them  give  no  Millon 's  reaction.  They  are  precipitated 
by  alkaloidal  reagents,  and  fairly  well  by  neutral  salts.  Th«y 
give  precipitates  with  ammoniacal  protein  solutions. 

Conjugated  Proteins 

These  compounds  are  made  up  of  protein  combined  with  some 
other  substance  or  substances.  The  non-protein  portion  is  called 
the  prosthetic  group : 

1.  Glycoproteins. — These  proteins  are  characterized  by  a 
high  content  of  a  carbohydrate  derivative,  usually  glucosamine. 
They  usually  are  divided  into  two  groups,  mucoids  from  the  tis- 
sues and  mucins  from  the  fluids  and  secretions  of  the  body.  As 
these  substances  are  extremely  difficult  to  purify  even  approxi- 
mately, there  is  much  disagreement  as  to  their  composition  and 
properties. 

(a)  Mucoids  or  Cliondroproteins. — The  mucoid  obtained  by 
extracting  the  ligament  and  tendon  in  the  preparation  of  albu- 
minoids may  be  studied  if  time  permits.  The  mucoid  is  precipi- 
tated by  acidifying  with  acetic  acid.  It  gives  the  usual  protein 
color  tests.  By  hydrolizing  with  10%  HCl  it  may  be  split  up. 
Sulphate  and  carbohydrate  may  be  detected  in  the  hydrolyzed 
liquid. 

(b)  Mucin. 

i.  Rinse  the  mouth  carefully  with  distilled  water,  and  collect 
a  test  tube  of  saliva.  If  there  appear  to  be  solid  particles  in  the 
liquid  it  should  be  filtered.  Add  10%  acetic  acid  as  long  as  a 
precipitate  forms.     This  precipitate  is  mucin.    It  is  not  soluble 


PROTEINS  243 

in  excess  of  the  acid.  Allow  to  settle,  decant  most  of  the  liquid, 
and  if  the  precipitate  is  sufficiently  heavy,  filter  and  use  small 
portions  of  the  residue  for  the  following  tests.  If  the  precipitate 
is  very  slight,  use  portions  of  the  saliva  containing  the  pre- 
cipitated mucin  from  which  most  of  the  clear  supernatant  liquid 
has  been  poured  off. 

ii.  On  a  portion  of  the  mucin  suspended  in  water  try  the 
biuret  and  the  xanthoproteic  tests.    The  tests  should  be  positive. 

iii.  Hydrolyze  a  portion  of  the  mucin  by  boiling  with  dilute 
HCl,  and,  with  portions  of  the  liquid,  test  for  sulphate,  and  for 
carbohydrate.    The  tests  should  be  positive. 

2.  Hemoglobins. — The  hemoglobins  are  compounds  consist- 
ing of  a  protein  combined  with  hemochromogen  or  some  similar 
substance.  In  connection  with  the  study  of  hemoglobin,  some 
other  constituents  of  blood  wall  be  considered.  Recall  the  inor- 
ganic materials  present  in  blood  as  determined  in  an  earlier 
chapter.  What  proteins  have  been  observed  in  the  blood  earlier 
in  this  chapter?  In  blood  are  also  found  fat,  sugar,  extractives, 
protein  decomposition  products  and  various  other  substances. 

i.  Spectroscopic  behavior  of  hemoglobin.  Study  the  spectro- 
scopic behavior  of  hemoglobin  and  its  derivatives  as  follows,  two 
students  working  together. 

(a)  Oxygenation  of  Hemoglohin. — The  color  of  oxyhemoglobin 
is  a  much  lighter  and  more  brilliant  red  than  that  of  hemoglobin. 
Dilute  defibrinated  blood  with  5  volumes  of  water.  The  blood 
is  ' '  laked, ' '  that  is,  the  hemoglobin  leaves  the  corpuscles  and  goes 
into  solution  in  the  water.  The  liquid,  which  was  opaque,  be- 
comes clear.  Laking  may  be  brought  about  in  various  other 
ways,  as  by  the  addition  of  a  small  amount  of  ether,  toluol,  etc. 
Shake  up  the  laked  blood  Avith  air,  closing  the  tube  with  the 
thumb.  The  color  becomes  bright  red,  as  the  hemoglobin  is 
changed  into  the  brighter  colored  oxyhemoglobin. 

Prepare  three  test  tubes  of  this  diluted  blood.  To  two  of 
them  add  Stokes'  fluid.  (This  is  a  mild  reducing  agent,  which 
contains  2%  Fe  SO.i,  3%  tartaric  acid,  and  ammonia  in  amount 
sufficient  to  redissolve  the  precipitate  which  forms  on  first  adding 


244 


PHYSIOLOGICAL    CHEMISTRY 


this  reagent.  The  reagent  must  be  freshly  prepared.)  The  color 
becomes  darker  red.  The  oxyhemoglobin  has  been  reduced  to 
hemoglobin.  Pour  the  blood  in  one  of  the  Stokes'  reagent  tubes 
several  times  from  one  test  tube  to  another.  The  brighter  oxy- 
hemoglobin color  reappears.     Evidently  hemoglobin  and  cxy- 


D 


E  i 


. 

1 

1 

■■I 

llll 

1 

;i  iiii; 
1!  ;;!!; 

1 

1 

i! ! 

1 

,            i 

5. 
6 

7 


Fig.  2.- — Absorption  Spectra. 

1.  Spectrum  of  sunlight  showing  Fraunhofer  lines. 

2.  Oxyhemoglobin   (0.37%). 

3.  Hemoglobin. 

4.  CO-hemoglobin. 

5.  Methemoglobin. 

6.  Acid  hematin  (in  ether). 

7.  Hemochromogen   (reduced  alkaline  hematin). 

8.  Acid  hematoporphyrin. 

hemoglobin  are  readily  converted  one  into  the  other.  Consider 
this  property  in  connection  with  the  role  which  hemoglobin  plays 
in  the  organism. 


PROTEINS  245 

(b)  Spectroscopic  Study  of  Blood  Pigment. — By  means  of  a 
spectroscope  observe  the  spectrum  produced  by  a  luminous  gas 
flame  and  by  electric  light.  The  spectrum  is  continuous.  Ob- 
serve also  the  incomplete  spectrum  of  the  non-luminous  bunsen 
flame.  Introduce  a  small  amount  of  a  sodium  compound  into  the 
flame  and  observe  the  yellow  band.  Such  a  spectrum  is  called  a 
bright  line  spectrum  and  the  particular  bands  observed  are  char- 
acteristic of  sodium.  Observe  the  spectrum  produced  by  sunlight. 
Notice  the  fine  dark  lines  at  various  intervals.  These  are  the 
Fraunhofer  lines,  and  the  most  prominent  of  them  will  be  used 
to  orient  the  absorption  bands  produced  by  the  hemoglobin  solu- 
tions examined.  In  making  a  spectroscopic  examination  and  in 
charting  the  result,  have  the  red  end  of  the  spectrum  always  on 
the  left.  This  avoids  confusion  and  error  in  interpreting  notes. 
Observe  and  chart  the  following  lines  in  your  notebook,  and  in- 
dicate them  on  all  your  spectrum  records.  From  left  to  right 
(beginning  in  the  red)  B  and  C,  two  prominent  lines  in  the  red ; 
D,  a  prominent  line  in  the  yellow  (really  two  lines  if  observed 
with  a  delicate  spectroscope) .  This  line  corresponds  to  the  bright 
lines  observed  above  in  the  sodium  spectrum;  E  and  b,  two 
prominent  lines  in  the  green ;  F  at  the  beginning  of  the  purple. 

(c)  Examine  oxyhemoglobin  solutions  of  various  concentra- 
tions spectroscopically  as  directed  below.  Blood  contains  be- 
tween 13%  and  14%  hemoglobin  so  the  percentage  concentra- 
tion of  hemoglobin  may  be  calculated  for  any  dilution.  The 
observations  are  made  through  a  flat  sided  cell.  Each  successive 
dilution  may  be  made  by  adding  an  equal  volume  of  distilled 
water  to  any  volume  of  the  liquid  to  be  diluted.  Make  observa- 
tions on  blood  in  the  following  dilutions :  Blood  shaken  with  air 
and  diluted  10  times,  20,  40,  80,  160,  320,  640  and  1280  times. 
If  the  solution  at  a  dilution  of  1280  still  shows  two  absorption 
bands,  dilute  further  until  only  one  band  remains.  Eecord 
results. 

(d)  Spectrum  of  Hemoglobin. — Pour  blood  diluted  40  times 
into  the  cell,  reduce  it  by  adding  a  small  amount  of  Stokes'  rea- 


246  PHYSIOLOGICAL    CHEMISTRY 

gent  and  observe  the  spectrum.     Record.     Repeat  with  blood 
diluted  80  and  160  times. 

(e)  Carbon  Monoxide  Hemoglobin. — Prepare  a  solution  of 
carbon  monoxide  hemoglobin  by  diluting  blood  160  times,  and,  in 
the  hood,  passing  illuminating  gas  through  the  liquid.  Observe 
the  cherry  red  color.  Observe  the  solution  spectroscopically  and 
chart  it,  comparing  the  location  of  the  bands  carefully  with  those 
given  by  an  oxyhemoglobin  solution.  To  a  little  of  the  carbon 
monoxide  hemoglobin  solution  add  Stokes '  reagent.  Observe  that 
the  spectrum  does  not  change,  as  did  that  of  oxyhemoglobin, 
which  it  resembles,  under  similar  conditions. 

Although  carbon  monoxide  forms,  a  fairly  stable  compound 
with  hemoglobin,  still  it  is  possible  to  remove  the  carbon  mon- 
oxide by  passing  a  brisk  air  stream  through  the  solution  for  some 
time.  The  solution  will  contain  oxyhemoglobin,  as  can  be  dem- 
onstrated by  reducing  with  Stokes'  reagent.  The  oxyhemoglobin 
is  reduced  to  hemoglobin. 

(f)  Metliemoglohin. — To  a  small  volume  of  blood  diluted  ten 
times  add  a  few  drops  of  a  fresh  solution  of  potassium  ferricya- 
nide.  Methemoglobin  is  formed.  The  color  becomes  a  dirty 
brown.  Examine  the  solution  with  the  spectroscope,  diluting 
somewhat  if  it  is  too  opaque,  and  chart.  While  one  student  is 
observing  the  spectrum,  let  his  partner  add  Stokes'  fluid  to  the 
liquid  in  the  cell.  Observe  that  the  methemoglobin  spectrum 
gives  way  to  that  of  oxyhemoglobin,  which  in  turn  is  replaced  by 
that  of  hemoglobin.  The  reducing  agent  first  changes  methemo- 
globin back  into  oxyhemoglobin  (these  two  compounds  contain 
the  same  amount  of  oxygen)  from  which  the  oxygen  is  then  re- 
moved by  the  reducing  agent,  hemoglobin  being  produced. 

(g)  Acid  Jiematin  may  be  prepared  by  treating  defibrinated 
blood  with  half  its  volume  of  glacial  acetic  acid  and  an  equal 
volume  of  ether.  The  liberated  hematin  dissolves  in  the  ether, 
and  this  solution  may  be  used  for  a  spectroscopic  examination. 
It  shows  a  distinct  band  between  C  and  D,  somewhat  nearer  C 
than  the  band  in  the  methemoglobin  spectrum.  A  second  fainter 
band  appears  between  D  and  F.    On  dilution  this  band  divides 


PROTEINS  247 

into  two,  a  broad  dark  band  in  the  green  between  b  and  F,  and  a 
narrow  fainter  band  to  the  left  of  E.  A  fourth  band  may  appear 
on  the  violet  side  of  D. 

(h)  HemocJiromogen. — (Reduced  alkaline  Hematin).  To 
blood  diluted  1-5  add  half  its  volume  of  10%  sodium  hydrate,  and 
warm  almost  to  boiling.  The  liquid  now  contains  alkaline 
hematin,  which  shows  a  single  absorption  band,  usually  rather 
faint,  lying  across  D,  and  mainly  toward  the  red  end  of  the  spec- 
trum. To  this  alkaline  hematin  solution,  add  Stokes'  reagent. 
The  liquid  now  contains  hemochromogen.  Chart  the  spectrum. 
The  formation  of  hemochromogen  is  a  very  delicate  test  for  blood 
stains,  as  solutions  of  hemoglobin  too  dilute  to  give  a  character- 
istic spectrum,  will  give,  upon  conversion  into  hemochromogen  by 
boiling  with  1%  sodium  hydrate  and  reduction  with  Stokes'  rea- 
gent, a  single  band  in  the  green  near  the  yellow. 

(i)  Acid  HematoporpJiyrin. — To  5  c.c.  of  concentrated  sul- 
phuric acid  in  a  test  tube  add  2-3  drops  of  undiluted  defibrinated 
blood,  mixing  well  after  the  addition  of  each  drop.  Observe  the 
color  and  chart  the  spectrum.  If  the  color  is  too  dark,  dilute 
with  glacial  acetic  acid. 

ii.  Crystallization  of  Blood  Pigment. 

(a)  Hemoglohiyi  Crystals. — Place  a  drop  of  defibrinated  rat 
or  guinea  pig  blood  on  a  slide,  add  an  equal  volume  of  water  and 
1-2  drops  ether.  Mix  and  cover.  In  a  few  minutes  crystals  of 
oxyhemoglobin  separate  out.  Observe  under  the  microscope  and 
draw. 

iii.  Cliemical  Tests  for  Becognizing  tJie  Presence  of  Blood. 

(a)  Hemin  Crystals. — Place  a  drop  or  two  of  blood  on  a  slide. 
Add  one  or  two  crystals  of  sodium  chloride  (no  more)  and  rub 
with  a  glass  rod  until  the  salt  has  dissolved.  Place  the  slide  on 
a  ring  about  a  foot  above  a  small  flame  and  allow  the  blood  to 
evaporate  slowly  to  complete  dryness.  Rub  the  red  residue  to  a 
powder  with  a  knife  blade,  collect  the  powder  in  a  small  pile  and 
add  a  drop  of  glacial  acetic  acid  from  a  glass  rod.  Rub  to  a 
paste,  place  a  portion  on  a  clean  slide,  and  add  a  drop  of  glacial 
acetic  acid.    Cover  with  a  cover  slip  and  cautiousl}^  heat  over  a 


248  PHYSIOLOGICAL    CHEMISTRY 

small  flame  until  the  acid  begins  to  boil.  Let  a  di^op  more  of  the 
acid  run  under  the  slide  and  allow  to  cool.  Examine  the  crystals 
of  hemin  (hematin  hydrochloride)  under  the  microscope  and 
draw.    The  crystals  are  brownish-red  rhomboids. 

This  test  is  one  of  the  best  methods  for  detecting  small  quan- 
tities of  blood  in  blood  stains.  Blood  stains  on  cloth,  etc.,  are 
soaked  in  distilled  water  or  alkali,  the  solution  evaporated  and 
treated  as  above.  The  formation  of  hemin  crystals  is  an  abso- 
lute proof  of  the  presence  of  blood;  it  does  not,  however,  dis- 
tinguish between  the  blood  of  man  and  that  of  some  other  ani- 
mals. 

(b)  Benzidene  Reaction. — The  benzidene  reaction  is  also  one 
of  the  most  delicate  and  reliable  tests  for  blood.  Different  solu- 
tions of  benzidene  vary  considerably  in  sensitiveness,  so  that  in 
practice  a  control  ahvays  should  be  run,  using  distilled  water  in 
place  of  blood,  Benzidene  solutions  should  be  kept  in  the  dark 
as  they  are  easily  altered  by  the  action  of  light.  Mix  a  saturated 
solution  of  benzidene  in  alcohol  or  glacial  acetic  acid,  with  an 
equal  volume  of  3%  hydrogen  peroxide  and  add  one  cubic  centi- 
meter of  a  dilute  blood  solution.  If  the  mixture  is  not  already 
acid,  acidify  with  acetic  acid.  Note  the  greenish  blue  color.  This 
test  may  be  performed  by  adding  a  small  amount  of  solid  benzi- 
dene, and  glacial  acetic  acid  instead  of  the  benzidene  solution. 

(c)  Guaiac  test.  This  test,  if  properly  performed  is  extremely 
delicate.  As  it  is  given  by  substances  other  than  blood,  however, 
conclusions  from  a  positive  result  should  be  drawn  only  with 
caution.  A  negative  result  is  conclusive  evidence  of  the  ab- 
sence of  blood. 

(1)  Dilute  defibrinated  blood  by  adding  2-3  drops  to  a  test 
tube  of  distilled  water.  Add  about  one-eighth  volume  of  hydro- 
gen peroxide  (3  vols,  per  cent)  and  float  on  the  surface  a  layer 
of  tincture  of  guaiacum  (or  of  guaiaconic  acid).  Note  the  slow 
appearance  of  a  green-blue  color  above  the  junction  of  the 
liquids.  Boil  a  small  volume  of  blood  and  repeat  the  test.  It  is 
still  positive. 

(2)  On  a  slice  of  raw  carrot  put  a  little  hydrogen  peroxide  and 


PROTEINS  249 

some  of  the  guaiacum  solution.  Observe  the  color.  Repeat  with 
boiled  carrot.  The  color  does  not  form.  The  reaction  also  is 
given  by  milk,  pus,  saliva,  and  various  other  substances,  but 
these  substances  do  not  give  the  test  after  having  been  boiled, 
thus  differing  from  blood.  They  contain  an  oxidase  which  is  re- 
sponsible for  the  reaction  and  which  is  destroyed  by  boiling.  In 
the  case  of  blood,  the  reaction  depends  probably  on  the  catalytic 
action  of  the  iron  constituent  of  heraatin, 

(d)  Catalase.  The  presence  of  a  catalase  in  blood  may  be  dem- 
onstrated readily.  To  a  little  defibrinated  blood  add  hydrogen 
peroxide.  Observe  the  bubbles  of  oxygen  given  off.  This  reac- 
tion is  due  also  in  some  measure  to  the  blood  pigment,  but  fresh 
blood  contains  a  catalase  in  addition.  Repeat  the  experiment 
with  boiled  blood.  The  test  is  negative,  as  the  catalase  is  de- 
stroyed by  boiling. 

(e)  (1)  In  testing  a  blood  stain,  in  addition  to  the  tests  given 
above,  (a,  b  and  c)  a  small  portion  of  the  fabric  should  be  ex- 
tracted with  glycerol  or  0.9%  sodium  chloride,  and  the  extract 
examined  with  the  microscope  for  corpuscles. 

(2)  An  aqueous  extract  of  the  stain  may  be  tested  for  the 
formation  of  hemochromogen.  If  the  stain  does  not  dissolve  in 
water  it  may  be  extracted  with  acidified  alcohol,  and  the  extract 
examined  with  the  spectroscope  for  acid  hematin. 

(f)  The  identification  of  a  stain  as  human  blood  is  accom- 
plished by  none  of  the  above  reactions.  The  final  proof  that  a 
blood  stain  has  come  from  a  human  subject  is  obtained  by  agglu- 
tination and  hemolysis  tests. 

iv.  The  globin  constituent  of  hemoglobin  may  be  demon- 
strated as  follows :  Corpuscles  are  washed  2  or  3  times  with  iso- 
tonic salt  solution,  the  corpuscles  being  separated  from  the 
liquid  each  tim.e  by  centrifuging.  A  small  quantity  of  the 
washed  corpuscles  is  then  placed  in  a  test  tube,  a  small  amount  of 
alcohol  and  of  hydrochloric  acid  added  and  the  mixture  heated 
on  the  water  bath.  The  liquid  turns  a  dark  brown  color  due  to 
the  formation  of  acid  hematin,  and  a  precipitate  forms  which 


250  PHYSIOLOGICAL    CHEMISTRY 

consists  of  the  globin.  This  may  be  filtered  off,  and  it  will  be 
found  to  give  the  usual  protein  color  tests. 

V.  Hemoglobin,  although  crystallizing  readily,  differs  from 
other  crystalloids  in  not  diffusing  through  an  animal  membrane. 

3.  Phosphoproteins.— The  phosphoproteins  are  compounds 
consisting  of  a  protein  combined  with  some  phosphorus-contain- 
ing substance  other  than  nucleic  acid  or  lecithin.  These  proteins 
often  are  called  nucleoalbumins ;  they  differ  from  the  nucleopro- 
teins  by  containing  no  purine  bases.  Two  members  of  the  group 
will  be  studied,  casein  from  milk  and  vitellin  from  egg  yolk.  In 
connection  with  these  two  substances,  some  other  constituents  of 
milk  and  egg  yolk  will  be  considered. 

i.  Caseinogen. 

(a)  Test  with  litmus  the  reaction  of  the  milk  furnished. 
Fresh  milk  is  neutral  or  faintly  alkaline. 

(b)  Boil  a  few  cubic  centimeters  of  milk.  Observe  that  the 
caseinogen  does  not  coagulate.  In  this  respect  caseinogen  differs 
from  most  other  proteins.  If  a  skin  forms  over  the  surface  of  the 
milk,  it  is  due  partly  to  evaporation  from  the  surface  of  the 
liquid,  partly,  perhaps,  to  coagulation  of  other  milk  proteins. 

(c)  To  a  few  cubic  centimeters  of  milk  add  an  equal  volume 
of  saturated  ammonium  sulphate.  The  precipitate  consists  main- 
ly of  caseinogen.  In  this  respect  caseinogen  resembles  the  glob- 
ulins. The  member  of  this  group  occurring  in  milk  also  is  pre- 
cipitated with  caseinogen,  but  its  amount  is  extremely  small. 
Filter  the  mixture.  Divide  the  filtrate  from  the  caseinogen  into 
two  portions.  Heat  one  to  boiling,  acidifying  slightly  if  neces- 
sary. The  slight  precipitate  is  lactalbumin.  Saturate  the  sec- 
ond portion  with  ammonium  sulphate.    Albumin  is  precipitated. 

(d)  The  usual  method  for  the  preparation  of  caseinogen  con- 
sists in  precipitating  with  dilute  acetic  acid.  This  process  is 
analogous  to  the  clotting  of  sour  milk ;  by  the  action  of  bacteria 
milk  sugar  is  fermented.  The  resulting  lactic  acid,  when  present 
in  sufficient  concentration,  causes  the  caseinogen  to  precipitate. 
Dilute  25  c.c.  of  milk  with  three  times  its  volume  of  water,  warm 
slightly  (about  to  body  temperature)   and  add  1%  acetic  acid 


PROTEINS  251 

drop  by  drop,  stirring  and  allowing  a  short  interval  to  elapse 
between  the  addition  of  successive  drops.  Caseinogen  flocks  out 
as  a  heavy  white  precipitate.  Add  acid  until  the  supernatant 
liquid  is  clear.  Filter  and  save  both  the  filtrate  (x)  for  use  in 
(e)  and  the  precipitate.  Dissolve  the  precipitate  in  2%  sodium 
carbonate,  and  reprecipitate  with  acetic  acid.  This  reprecipita- 
tion  is  for  the  purpose  of  freeing  the  caseinogen  from  fat,  which 
is  carried  down  mechanically.  It  may  be  repeated  several  times 
if  a  purer  product  is  desired.  Redissolve  the  caseinogen  in  2% 
sodium  carbonate,  filter  through  a  wet  filter  to  remove  more  of 
the  fat,  and  with  this  solution  perform  the  biuret,  Millon,  and 
xanthoproteic  tests,  and  the  test  for  loosely  combined  sulphur. 
All  should  be  positive.  The  presence  of  phosphorus  in  caseino- 
gen already  has  been  observed. 

(e)  The  filtrate  (x)  from  the  caseinogen  prepared  in  (d)  con- 
tains lactalbumin  and  lactoglobulin,  whose  presence  in  milk  was 
demonstrated  in  (c).  The  filtrate  (x)  is  already  acid.  Boil  it 
and  add  2%  sodium  carbonate  drop  by  drop  until  nearly  neutral. 
If  the  liquid  becomes  alkaline,  reacidify  with  a  few  drops  of 
acetic  acid.  This  treatment  removes  lactalbumin  and  lactoglobu- 
lin. Filter  from  the  precipitate  and  test  the  filtrate  with  Feh- 
ling'g  solution  for  sugar,  and  for  phosphates.  Casein  is  one  of 
the  chief  constituents  of  cheese,  which  also  contains  much  fat. 

(f )  Recall  that  unboiled  milk  gives  a  positive  guaiac  test. 

ii.  Vitellin.  This  phosphoprotein  is  found  in  egg  yolk.  Ex- 
tracting the  yolk  with  ether  removes  the  greater  part  of  the 
lecithin  and  cholesterol.  Vitellin  is  soluble  in  10%  NaCl.  If  the 
solution  is  poured  into  a  large  beaker  of  distilled  water  the  vitel- 
lin precipitates.  Vitellin  responds  to  the  protein  color  tests  and 
it  may  be  shown  to  contain  phosphorus ;  on  digestion  with  arti- 
ficial gastric  juice,  the  phosphoproteins  leave  an  insoluble  resid^^e. 
This  residue  consists  of  pseudonuclein,  which  contains  phos- 
phorus. 

4.  Nncleoproteins.- — Nucleoproteins  are  present  in  all  cells. 
They  may  be  prepared  from  various  organs  and  tissues. 

i.  Preparation  of  nucleoprotein  from  the  pancreas. 


252  PHYSIOLOGICAL    CHEMISTRY 

Hammarsten's  MetJiod. — Grind  a  piece  of  fresh  beef  pancreas 
in  the  meat  grinder,  throw  the  pulp  into  200-300  c.c.  of  hot 
water,  and  boil  for  about  ten  minutes.  Filter  hot.  The  filtrate 
will  be  pale  yellow  and  fairly  clear.  Cool  under  the  tap,  and 
acidify  with  sufficient  acetic  acid  to  make  the  concentration  of 
acid  0.5-1.0%.  The  nucleoprotein  is  precipitated  and  quickly 
settles  to  the  bottom.  What  other  groups  of  proteins  are  pre- 
cipitated in  this  way?  Filter  off  the  precipitate,  suspend  it  in 
200  c.c.  distilled  water,  and  add  ammonia  cautiously  until  the 
precipitate  has  just  dissolved.  Eeprecipitate  the  nucleoprotein 
with' acetic  acid  as  above.  If  a  pure  product  is  desired,  the  proc- 
ess of  dissolving  in  alkali  and  reprecipitation  with  acid  should  be 
repeated  several  times.  The  precipitate  is  filtered  off,  washed 
with  water  containing  a  few  drops  of  acetic  acid,  and  then  with 
about  50  c.c.  of  hot  alcohol  in  small  portions  (be  careful  of  fire) . 
Spread  the  washed  nucleoprotein  upon  a  carefully  cleaned  spot 
on  a  tile,  and  manipulate  it  to  remove  the  alcohol. 

ii.  With  small  portions  of  the  nucleoprotein  prepared  in  (i) 
perform  the  Millon,  biuret,  and  xanthoproteic  tests.  All  are 
positive. 

iii.  Note  that  nucleoprotein  was  not  coagulated  by  boiling. 

iv.  Fuse  a  small  portion  of  nucleoprotein  with  fusion  mixture, 
dissolve  the  residue  in  dilute  nitric  acid  and  test  for  phosphorus 
and  iron.    Both  tests  should  be  positive. 

V.  Cover  a  small  portion  of  nucleoprotein  with  pepsin  solu- 
tion, and  incubate  at  least  24  hours.  Note  the  undigested 
residue  of  nuclein. 

vi.  Hydrolysis  of  nucleoprotein.  Mix  up  the  remainder  of  the 
nucleoprotein  with  10  times  its  volume  of  5%  hydrochloric  acid, 
and  boil  in  the  hood  for  about  half  an  hour.  To  identify  the  de- 
composition products  of  nucleoprotein  divide  the  liquid  into  four 
parts  and  make  the  following  tests :  For  sugar  with  Fehling  's 
test;  for  phosphate;  for  purine  bases.  To  detect  purine  bases, 
add  an  excess  of  ammonia,  and  then  a  small  amount  of  silver 
nitrate.  Under  these  conditions  purine  bases  are  precipitated  as 
their  silver  salts. 


PROTEINS  253 

vii.  Glycoproteins  and  many  phosphoproteins  also  are  pre- 
cipitated by  acetic  acid  and  dissolve  in  dilute  alkalies.  From 
the  above  experiments,  devise  a  way  to  distinguish  among  gly- 
coproteins, phosphoproteins  and  nucleoproteins. 

5.  Lecithoproteins. — This  group  of  conjugated  proteins  has 
not  been  exhaustively  studied.  It  includes  compounds  of  simple 
protein  with  the  lecithins,  the  substances  being  known  as  leci- 
thans,  and  also  with  some  other  members  of  the  phosphatid 
group. 

Derived  Proteins 

1.  Primary  Protein  Derivatives. — 

(a)  Proteans. — These  protein  derivatives  are  formed  by  the 
action  of  very  small  quantities  of  acids,  of  water  or  of  enzymes 
on  most  of  the  proteins.  They  are  characterized  chiefly  by  al- 
tered solubilities,  as  little  is  known  of  the  proteans. 

(b)  Metaproteins. — By  the  further  action  of  weak  acids,  or  of 
alkalies,  products  are  formed  which  are  readily  soluble  in  weak 
acids  or  alkalies,  insoluble,  however,  in  neutral  solution.  The 
metaproteins  are  divided  into  two  classes  according  to  the  manner 
of  their  preparation.  These  substances  often  are  called  albu- 
minates. 

i.  Acid  Metaprotein. 

Measure  25  c.c.  of  egg  albumin  solution  (the  solution  furnished 
will  be  Q^g  white  diluted  1  to  10)  into  a  beaker,  add  an  equal 
volume  of  0.4%  hydrochloric  acid  and  heat  on  a  water  bath  at 
40°-50°  for  about  Y2  hour.  While  waiting,  the  preparation  of 
alkali  metaprotein  may  be  started,  if  time  permits  the  study  of 
both  of  these  substances.  At  the  end  of  the  heating  period,  boil 
the  solution  vigorously.  This  will  coagulate  any  unchanged 
albumin.  The  metaprotein  is  not  coagulated  by  boiling  in  acid 
solution.     Filter  if  necessary. 

Exactly  neutralize  the  solution  of  acid  albuminate  with  0.4% 
sodium  hydrate.  A  white  precipitate  should  form.  This  will 
redissolve  in  a  slight  excess  of  alkali,  so  that  the  process  of 
neutralization  must  be  carried  on  with  great  care.     Filter  off 


254  PHYSIOLOGICAL    CHEMISTRY 

the  precipitate  and  wash  it  once  with  distilled  water.  Suspend 
the  precipitate  in  a  small  amount  of  distilled  water,  and  with 
small  portions  make  the  following  tests : 

(1)  Test  for  loosely  combined  sulphur.  The  result  should  be 
positive. 

(2)  Add  a  small  amount  of  0.4%  hydrochloric  acid.  The 
precipitate  dissolves.  Neutralize  carefully  with  sodium  carbon- 
ate.    The  metaprotein  is  precipitated. 

(3)  Add  a  small  amount  of  0.4%  NaOH.  The  metaprotein 
dissolves.    Neutralize  with  0.4%  HCl.    It  precipitates. 

(4)  Metaprotein  may  be  distinguished  from  globulins  by  the 
fact  that  it  is  insoluble  in  ammonium  sulphate  of  any  concentra- 
tion, whereas  globulins  dissolve  in  ammonium  sulphate  of  con- 
centrations less  than  half  saturation,  and  are  precipitated  only 
at  this  latter  concentration. 

(5)  Boil  a  small  portion  of  the  neutral  suspension.  Cool  and 
add  0.4%  HCl.  The  metaprotein  no  longer  dissolves  in  dilute 
acid, — it  has  been  coagulated  by  boiling  in  neutral  solution.  Re- 
call that  it  is  not  coagulated  by  boiling  in  acid  or  alkaline  solu- 
tion. 

(6)  Dissolve  the  remainder  of  the  acid  albuminate  in  0.4% 
HCl.  Apply  (i)  the  biuret  and  Millon  tests.  Results  should  be 
positive,  (ii)  Try  precipitation  with  mercuric  chloride.  Acid 
metaprotein  made  from  meat  is  known  as  syntonin. 

Acid  metaprotein  is  especially  interesting  from  the  fact  that 
it  is  the  first  product  formed  in  the  digestion  of  proteins  in  the 
stomach  by  the  acid  gastric  juice. 

ii.  Alkali  metaprotein. — Although  differing  in  some  important 
respects,  many  of  the  properties  of  alkali  metaprotein  are  similar 
to  those  of  acid  metaproteins.  If  time  permits,  it  may  be  pre- 
pared in  the  same  manner  as  acid  metaproteins,  using  0.4% 
sodium  hydroxide  in  place  of  the  acid.  Its  properties  are  similar 
to  those  of  acid  metaprotein,  with  the  exception  that  it  gives  a 
less  definite  neutral  sulphur  test,  as  a  portion  of  the  sulphur  is 
split  off  in  its  manufacture. 

(c)   Coagulated  Proteins. — These  protein  derivatives  are  pro- 


PROTEINS  255 

duced  from  proteins  by  the  action  of  heat,  by  standing  under 
alcohol,  by  the  action  of  certain  enzymes,  and  in  various  other 
ways.  Much  of  the  protein  material  of  our  food  is  coagulated 
by  cooking  before  ingestion. 

i.  Measure  about  10  c.e.  of  imdiluted  egg  white  into  a  thin- 
walled  test  tube  and  immerse  the  tube  in  boiling  water  for  6-8 
minutes.  Observe  the  progress  of  coagulation.  Remove  the  coag- 
ulated protein  from  the  tube  by  means  of  a  glass  rod,  or  the  wire 
handle  of  a  test  tube  brush. 

(1)  With  small  pieces,  test  its  solubility  in  water  and  10% 
NaCl. 

(2)  In  each  of  two  test  tubes  put  small  pieces  of  the  egg  white. 
To  one  add  0.5%  NaOH,  to  the  other  0.2%  HCl.  Warm  to  40° 
for  some  time.  Observe  that  solution  takes  place.  Neutralize 
each  carefully.  The  precipitate  indicates  that  the  protein  was 
converted  into  metaprotein. 

(3)  Place  pieces  of  egg  white  in  each  of  two  test  tubes  and 
test  their  digestibility  by  pepsin  and  trypsin,  adding  a  drop  of 
chloroform  and  a  little  toluol  and  digesting  several  hours  in  the 
incubator.    Note  that  the  coagulated  albumin  is  readily  digestible. 

(4)  With  a  small  piece  of  egg  white  in  water  try  Millon's 
reaction  and  the  xanthoproteic  test.    Both  are  positive. 

(5)  Biuret  test.  Partly  dissolve  a  piece  of  egg  white  in  concen- 
trated NaOH.  Cautiously  add  very  dilute  copper  sulphate.  Ob- 
serve the  lavender  biuret  color.  From  the  above  results  it  ap- 
pears that  coagulated  albumin  still  belongs  to  the  protein  group. 
The  exact  nature  of  the  alterations  in  the  protein  molecule 
brought  about  by  coagulation  is  not  understood. 

2.  Secondary  Protein  Derivatives. 

(a)  Proteoses. — The  intermediary  products  of  protein  hydro- 
lysis are  somewhat  arbitrarily  subdivided  into  various  groups 
distinguished  by  the  concentration  of  ammonium  sulphate  neces- 
sary to  cause  their  precipitation  and  by  other  similar  character- 
istics. Products  which  are  not  precipitated  by  ammonium  sul- 
phate are  said  to  have  passed  beyond  the  proteose  state. 
"Witte's  peptone"  and  "Armour's  peptone"  are  in  reality  mix- 


256  PHYSIOLOGICAL    CHEMISTRY 

tures  of  proteoses  and  peptones.    The  former  is  mainly  proteose, 
the  latter  mainly  peptone. 

1.  Dissolve  about  a  half  teaspoonful  of  "Witte's  peptone"  in 
sufficient  water  to  cause  solution.  Boil  to  coagulate  any  un- 
changed protein.  Filter  if  necessary.  Saturate  the  hot  solution 
with  ammonium  sulphate.  Proteoses  are  precipitated.  Eemove 
the  precipitate  either  by  filtering,  or,  if  it  has  formed  in  clumps, 
by  collecting  with  a  glass  rod  or  with  a  small  watch  glass.  If  the 
latter  method  has  been  employed,  filter  the  liquid  to  remove  any 
remaining  proteose,  and  reserve  the  filtrate  for  the  study  of  pep- 
tones in  (b)  below.  Press  the  proteose  precipitate  between  filter 
papers  to  remove  as  much  sulphate  as  possible,  and  dissolve  in  a 
small  amount  of  water.  Add  solid  barium  carbonate  in  excess 
and  boil.  Filter  from  the  preciptated  barium  sulphate  and  use 
the  filtrate  which  contains  a  mixture  of  proteoses,  for  the  follow- 
ing tests : 

(1)  Biuret,  Millon,  Adamkiewiez. 

(2)  Precipitation  with  concentrated  HNO3.  If  a  precipitate 
forms,  it  consists  of  the  so-called  primary  proteoses,  the  only 
members  of  this  group  which  are  precipitated  by  nitric  acid. 
Performed  as  a  "ring  test,"  this  test  is  known  as  Heller's  ring 
test. 

(3)  Precipitate  with  picric  acid.  Warm  and  observe  that  the 
precipitate  dissolves.    Cool.    It  reappears. 

(4)  Note  that  proteoses  are  not  coagulated  by  boiling,  even  in 
acid  solution. 

(5)  The  lower  members  of  the  proteose  group  are  somewhat 
diffusible  through  an  animal  membrane. 

(b)  Peptones. — The  filtrate  from  proteoses  reserved  in  (a)  i. 
which  contains  peptones,  or  preferably  a  solution  of  Armour's 
peptone  may  be  examined  for  peptones.  After  removal  of  pro- 
teoses by  saturating  with  ammonium  sulphate,  concentrate  to  a 
small  volume,  cool,  pour  off  the  liquid  from  the  ammonium  sul- 
phate crystals,  and  boil  it  with  solid  barium  carbonate  until  sul- 
phate is  completely  removed.  Filter  and  test  the  filtrate  for  the 
properties  of  peptones. 


PROTEINS  257 

(1)  Perform  the  biuret,  Millon  and  Adamkiewicz  tests.  The 
biuret  gives  a  redder  color  than  with  proteins.  The  Millon  and 
Adamkiewicz  tests  are  usually  faint  or  negative,  since  tyrosin 
and  tryptophane  are  split  off  early  in  the  disintegration  of  pro- 
tein. 

(2)  Observe  that  peptones  are  not  precipitated  by  concen- 
trated nitric  acid. 

(3)  Try  precipitation  with  picric  acid,  and  with  potassium 
ferrocyanide  in  a  solution  made  acid  with  acetic  acid. 

(4)  Recall  that  peptones  are  not  coagulated  by  boiling. 

(5)  The  peptones  diffuse  through  an  animal  membrane  more 
readily  than  do  the  proteoses.  Since  proteins  do  not  diffuse,  a 
mixture  of  protein  and  peptones  may  be  freed  from  peptones  by 
dialysis. 

(e)  Peptids. — -Those  decomposition  products  of  the  proteins 
which  are  made  up  of  a  relatively  small  number  of  amino  acids 
are  known  as  peptids, — e.g.  di-,  tri-,  tetra-,  poly-peptids,  etc., 
according  to  the  number  of  amino  acids  making  up  the  peptid 
molecule.  The  study  of  this  group  of  protein  derivatives 
requires  an  amount  of  time  obtainable  only  in  a  more  specialized 
course. 

(d)  Amino  Acids. — The  detailed  study  of  these  final  products 
of  protein  hydrolysis,  as  with  the  peptids,  requires  more  time 
than  can  be  devoted  to  the  subject  in  a  general  course  in  bio- 
chemistry. Certain  members  of  the  group  will  be  studied,  how- 
ever. 

i.  The  student  should  be  furnished  with  a  mixture  of  protein 
decomposition  products  obtained  by  digesting  protein  material 
for  several  days  Avith  trypsin.  Test  the  liquid  with  the  biuret 
reaction.  The  result  will  give  an  idea  of  the  stage  to  which  the 
digestion  has  progressed.  Nearly  neutralize  100  e.c.  of  the  diges- 
tion mixture  with  dilute  hydrochloric  acid.  When  nearly  neutral, 
exactly  neutralize  with  0.2%  HCl  or  with  0.2%  sodium  carbonate 
to  pj-eeipitate  metaprotein.  Filter,  if  necessary  and  concentrate 
the  filtrate,  being  careful  to  make  the  solution  slightly  acid  with 
acetic  acid,  as  heating  in  alkaline  solution  will  cause  decomposi- 


258  PHYSIOLOGICAL    CHEMISTRY 

tion  of  the  amino  acids.  The  concentration  may  be  carried  on 
over  a  free  flame  or  wire  gauze  until  the  liquid  becomes  fairly 
concentrated.  The  process  then  should  be  continued  on  a  water 
bath.  To  the  resulting  syrupy  liquid,  add  alcohol  as  long  as  a 
precipitate  of  proteoses  and  peptones  forms.  Remove  as  much 
as  possible  of  this  sticky  precipitate,  warm  slightly,  and  filter. 
The  alcoholic  filtrate  contains  leucine  and  tyrosine.  Concentrate 
the  alcoholic  solution  on  the  water  bath  and  allow  it  to  stand  in 
a  cool  place  (over  night  if  possible).  Tyrosine,  being  the  more 
insoluble  of  the  two  amino  acids,  crystallizes  first.  Leucine  comes 
down  more  slowly.  When  a  good  crop  of  crystals  has  been 
obtained,  filter  them  off,  add  a  small  amount  of  water  and  warm 
gently.  Leucine  goes  into  solution  while  tyrosine  remains  undis- 
solved. Concentrate  the  leucin-filtrate  and  allow  it  to  stand 
(over  night  if  necessary). 

(1)  Tyrosine. 

(i)  Observe  the  crystal  form  with  the  microscope.  Tyrosine 
crystallizes  in  fine  needles,  which  often  group  into  rosettes  or 
sheaves.  If  the  crystals  are  not  well  formed,  add  a  drop  of  water, 
warm,  and  allow  to  cool  slowdy. 

(ii)  Observe  that  tyrosine  is  quite  insoluble  in  cold  water, 
but  much  more  soluble  in  hot. 

(iii)  Perform  Millon's,  test  with  a  small  amount  of  tyrosine. 
Recall  that  the  Millon  test  as  given  by  proteins  is  due  to  the 
tyrosine  which  they  contain. 

(2)  Leucine. 

(i)   Observe  the  crystals  under  the  microscope  (broad  plates), 
(ii)   Record  solubility  of  leucine  in  cold  water  and  hot  water. 

(3)  Salts  of  Atnino  Acids. — The  copper  salts  of  the  amino 
acids  are  extremely  serviceable  in  separating  these  compounds, 
as  their  solubilities  vary  considerably.  To  a  solution  of  either 
tyrosine  or  leucine,  preferably  the  former,  in  hot  water,  add  a 
few  drops  of  diluted  copper  sulphate  solution.  Observe  the  blue 
color  of  the  copper  salt  of  the  amino  acid.  These  salts  may  be 
prej^ared  by  boiling  the  amino  acid  with  a  neutral  suspension  of 
freshly  precepitated  copper  oxide. 


PROTEINS  259 

ii.  Preparation  of  Cysiin  from  Wool. — The  student  will  be 
furnished  with  a  mixture  resulting  from  the  hydrolysis  of  wool 
with  concentrated  hydrochloric  acid.  To  about  50  c.c.  of  the 
liquid  add  solid  sodium  acetate  until  congo  red  paper  no  longer 
indicates  an  acid  reaction.  This  indicator  is  blue  in  acid  solu- 
tion, red  in  alkaline.  A  precipitate  consisting  mainly  of  cystin 
is  thrown  down.  If  the  precipitate  is  not  heavy,  allow  the  mix- 
ture to  stand.  Filter  off  the  precipitate,  wash  with  a  little  cold 
water,  and  dissolve  in  a  small  volume  of  5%  hydrochloric  acid. 
If  the  solution  is  dark  colored,  boil  with  animal  charcoal  until  a 
small  filtered  portion  is  colorless  or  pale  yellow.  Filter,  and  to 
the  hot  solution  add  hot  sodium  acetate  solution.  The  cystin 
will  separate  out  in  crystal  form  as  large  pentagons,  hexagons 
or  plates. 

(1)  Examine  the  cystin  crystals  under  the  microscope. 

(2)  Dissolve  a  small  amount  of  cystin  in  caustic  soda,  add  lead 
acetate  and  boil.  Observe  the  positive  ''neutral  sulphur"  test 
and  recall  that  as  given  by  proteins,  this  test  depends  upon  their 
content  of  cystin  or  cystein. 


CHAPTER  VII 
SALIVAEY  DIGESTION 

Rinse  the  mouth  with  distilled  water  and  collect  some  saliva 
by  chewing  a  small  piece  of  paraffin.  Filter  and  note  color, 
and  transparency.  The  saliva  obtained  in  this  way  is  a  mix- 
ture of  the  secretions  of  the  three  classes  of  salivary  glands. 
Its  composition  varies  with  the  nature  of  the  stimulus  causing 
secretion. 

Note  the  turbidity  which  increases  on  standing  as  a  result 
of  the  precipitation  of  calcium  carbonate.  Saliva  usually  con- 
tains epithelial  cells  or  cell  debris.  Test  the  chemical  reaction 
of  the  saliva  with  phenolphthalein,  litmus,  and  methyl  orange. 
Saliva  is  usually  slightly  alkaline,  but  not  sufficiently  so  to 
turn  phenolphthalein  red.  This  indicator  turns  color  when 
the  hydrogen-ion  concentration  is  N  X  lO"''-  Saliva  usually 
is  alkaline  to  litmus,  which  changes  color  at  a  hydrogen-ion 
concentration  of  N  X  10'^  or  about  the  true  neutral  point. 
The  reaction  of  the  saliva  thus  lies  usually  between  a  hydro- 
gen-ion concentration  of  N  X  10'^  and  N  X  lO'^-  In  case 
the  saliva  reacts  acid  to  litmus,  it  still  will  be  alkaline  to 
methyl  orange,  which  changes  color  at  a  hydrogen-ion  con- 
centration of  N  X  10"*. 

I.  Composition. — 

a.  Mucin. — Recall  the  preparation  and  properties  of  mucin 
studied  in  the  work  on  proteins. 

b.  Other  Proteins. — The  saliva  contains,  in  addition  to  mucin, 
small  traces  of  other  proteins,  the  presence  of  which  may  be 
demonstrated  after  the  removal  of  mucin.  Precipitate  mucin 
by  adding  acetic  acid,  filter  and  apply  the  biuret  test  to  the 
filtrate. 

260 


SALIVARY   DIGESTION  261 

c.  1.  Test  saliva  for  chlorides  and  for  sulphates. 

2,  For  sulphoeyanates  as  follows:  To  2  c.c.  of  saliva  in  a 
small  evaporating  dish,  add  a  few  drops  of  dilute  HCl  and 
then  a  drop  or  two  of  dilute  ferric  chloride.  A  reddish  color 
indicates  the  presence  of  sulphocyanate.  Compare  the  color 
with  that  produced  by  adding  a  similar  amount  of  ferric 
chloride  to  distilled  water  and  HCl  in  an  evaporating  dish. 
Note  that  the  color  is  a  pure  greenish  yellow  with  no  sugges- 
tion of  pink.  To  this  control  add  a  few  drops  of  dilute  potas- 
sium sulphocyanate.  A  comparison  of  this  color  with  that 
obtained  with  saliva  will  indicate  the  very  small  amount  of 
sulphocyanate  in  the  latter  fluid. 

3.  Phosphates  and  carbonates  also  occur  in  the  saliva  and 
may  be  detected  by  appropriate  tests. 

d.  Ptyalin. — This  ferment  acts  on  starch,  breaking  it  down 
into  dextrins,  maltose,  and  isomaltose.  It  may  be  isolated  by 
precipitation  with  alcohol.  Its  action  may  be  studied,  how- 
ever, without  isolation.  Saliva  is  said  to  contain  an  erepsin, 
which  acts  on  peptids.  Its  action  is  unimportant,  however. 
Saliva  contains  no  lipase. 

II.  Dig-estive  Action. — 

a.  On  Starch. — Prepare  a  starch  mucilage  by  mixing  about 
one  gram  of  starch  with  25  c.c.  cold  distilled  water.  Add  about 
75  c.c.  of  boiling  water  and  boil  for  15  minutes,  stirring  occa- 
sionally and  adding  water  to  replace  loss  by  distillation.  Cool, 
and  use  this  mucilage  in  the  following  experiments. 

Arrange  6  test  tubes  as  follows: 

1.  6  c.c.  distilled  water  and  a  few  grains  of  raw  starch. 

2.  5  c.c.  water,  1  c.c.  saliva  and  a  few  grains  of  raw  starch. 

3.  1  c.c  saliva  and  5  c.c  starch  mucilage. 

4.  1  c.c.  saliva  and  5  c.c.  starch  mucilage  and  3  drops  10% 
NaOH. 

5.  1  c.c  saliva  and  5  c.c  starch  mucilage  and  5  drops  10% 
HCl. 


262  PHYSIOLOGICAL    CHEMISTRY 

6.  Boil  a  few  c.c.  of  saliva  thoroughly  for  three  or  four 
minutes,  cool,  and  add  1  c.c.  to  5  c.c.  starch  mucilage. 

Put  all  six  tubes  in  a  beaker  of  water  warmed  to  40°  for 
15  minutes.  Test  each  solution  for  sugar  with  Fehling's  solu- 
tion and  for  starch  with  iodine.  A  positive  Fehling  indi- 
cates that  a  portion  of  the  starch  has  been  broken  down 
into  a  reducing  sugar  (maltose  and  isomaltose).  If  the  iodine 
test  is  still  blue,  some  starch  remains.  If  it  is  reddish,  the  solu- 
tion contains  dextrine.  If  iodine  gives  no  color,  the  material 
has  all  passed  to  the  achroodextrin  stage  or  beyond. 

Record  the  results  of  your  observations  and  draw  conclu- 
sions as  to  the  conditions  under  which  ptyalin  will  digest  starch. 

7.  Effect  of  cooling. — Place  a  test  tube  containing  saliva  in 
a  freezing  mixture  for  a  few  minutes.  Cool  5  c.c.  of  starch 
mucilage  in  a  separate  test  tube.  Add  1  c.c.  of  the  saliva  to  5 
c.c.  starch  mucilage  and  leave  in  a  freezing  mixture  for  15 
minutes^  In  a  portion,  test  for  sugar  with  Fehling's  solution. 
If  the  solutions  have  been  well  cooled,  little  or  no  digestion  will 
have  taken  place, — at  least  much  less  than  at  body  temperature. 

The  most  favorable  reaction  for  ptyalin  digestion  is  a  very 
weak  acidity  (Hydrogen-ion  concentration  N  X  lO"*''^).  A 
hydrogen-ion  concentration  of  N  X  10'*  is  sufficient  acid  to 
stop  its  action.  It  will  act  in  a  weakly  alkaline  solution  such 
as  the  saliva,  however.  The  acidity  of  the  contents  of  tube 
number  5  above  should  be  sufficient  to  destroy  ptyalin. 

b.  On  Cane  Sugar. — Add  saliva  to  a  few  c.c.  of  cane  sugar 
solution.  Digest  at  40°  for  15  minutes  and  test  with  Fehling's 
solution.  No  reduction  should  occur,  since  saliva  contains  no 
invertase. 

c.  On  Proteins  and  Fats. — Proteins  and  fats  are  not  digested 
by  saliva,  since  this  secretion  contains  no  proteolytic  or  lipolytic 
enzyme.  The  erepsin  mentioned  above  is  believed  to  be  unim- 
portant, and  acts  not  on  proteins,  but  on  peptids. 

d.  Progress  of  Digestion  ly  Ptyalin. — To  50  c.c.  of  starch 
mucilage  add  10  c.c.  of  saliva.  Digest  at  40°  and  at  intervals  of 
a  minute  or  two,  remove  a  few  drops  and  test  with  iodine.    From 


SALIVARY   DIGESTION  263 

the  results  of  the  iodine  test,  record  the  time  required  in  each 
stage  of  the  digestion  of  starch  to  achroodextrin  under  the  above 
conditions. 

e.  Products  of  Ptyalin  Digestion. — The  dextrins  and  maltose 
produced  by  the  action  of  ptyalin  may  be  isolated  by  precipitat- 
ing the  former  with  alcohol.  In  the  remaining  solution  maltose 
may  be  identified  by  its. reaction  with  Fehling's  solution  and 
the  preparation  of  its  osazone  with  phenylhydrazine. 


CHAPTER  VIII 
GASTRIC  DIGESTION 

Preparation  of  Artificial  Gastric  Juice 

Strip  the  mucous  membrane  from  the  stomach  of  a  pig,  and 
cut  into  small  pieces.  Cut  about  %  of  the  mucous  membrane 
into  very  small  pieces,  place  in  a  small  beaker  and  cover  Avith 
glycerine.  Allow  to  stand  at  room  temperature  for  24  hours, 
stirring  occasionally.  Decant  or  draw  off  with  a  pipette  the 
resulting  glycerine  solution  of  pepsinogen.  This  solution  will 
keep  for  a  long  time.  Use  it  for  the  digestion  experiments 
in  this  chapter.  To  the  remaining  %  of  the  gastric  mucosa, 
add  300  c.c.  of  0.35%  HCl  (3  c.c.  of  concentrated  HCl  diluted 
to  300  c.c),  a  few  c.c.  of  chloroform  and  set  in  the  incubator  at 
40°  for  at  least  48  hours,  or  longer  if  possible.  The  extracted 
pepsin  will  digest  the  protein  of  the  mucosa,  and  the  mixture 
may  be  studied  for  the  products  of  peptic  digestion  as  described 
below. 

I.  Composition  of  Gastric  Juice. — 

a.  Natural  gastric  juice  contains  small  amounts  of  mucin 
and  other  proteins. 

b.  Inorganic  salts,— NaCl,  earthy  phosphates,  etc. 

c.  Acids,  chiefly  HCl.  The  acidity  of  the  stomach  contents 
may  be  due  to  several  factors,  e.  g.,  free  HCl,  organic  acids,  acid 
combined  with  protein,  and  acid  salts.  The  sum  of  these  is 
known  as  the  total  acidity.  It  may  be  determined  by  titrating 
with  N/10  NaOH,  using  phenolphthalein  as  indicator.  Until 
recently  the  individual  factors  which  go  to  make  up  total  acidity 
have  been  estimated  by  the  use  of  various  indicators  sensitive  to 
different  concentrations  of  hydrogen-ions.  These  methods 
have  been  shown  to  be  subject  to  great  inaccuracy. 

264 


GASTRIC    DIGESTION  265 

If  it  is  desired  to  know  the  hydrogen-ion  concentration  of 
gastric  contents,  the  determination  may  be  made  by  electrolytic 
methods,  or  approximately  by  the  use  of  certain  series  of 
indicators. 

For  most  clinical  work  the  only  data  required  are  total 
acidity  as  determined  by  titration,  using  phenolphthalein  as 
indicator,  and  free  hydrochloric  acid  as  determined  by  titration 
using  Toepfer's  reagent  as  indicator  or  as  determined  with 
Guenzberg's  reagent. 

1.  Study  the  following  indicators,  by  observing  their  color 
in  faintly  acid  and  faintly  alkaline  solution. 

(a)  Congo  red  gives  a  blue  color  with  free  HCl,  violet  with 
an  organic  acid  and  brown  with  combined  HCl.  Add  a  few 
drops  of  Congo  red  to  0.2%  HCl  and  to  0.5%  acetic  acid.  Neu- 
tralize one  with  NaOH.  Add  2-3  drops  of  congo  to  a  few  c.c. 
of  your  hydrochloric  acid  extract  of  pig's  stomach. 

(b)  Guenzberg's  reagent  (2  g.  phloroglucin,  1  g.  vanillin, 
100  c.c.  alcohol)  produces  a  purplish  red  color  on  evaporating 
with  free  HCl.  Place  2  drops  of  the  reagent  in  an  evaporating 
dish.  Evaporate  to  dryness  on  the  water  bath.  A  yellow  spot 
results.  Add  2  drops  of  0.2%  PICl  and  replace  on  the  water 
bath.  Observe  the  red  spot.  Repeat  with  2  drops  of  your  HCl 
extract  of  stomach  mucosa.  Repeat  with  2  drops  of  lactic  acid. 
Guenzberg's  test  may  be  made  roughly  quantitative  by  dilut- 
ing the  acid  solution  until  it  will  just  respond  with  a  faint 
positive  reaction.  At  this  dilution  the  HCl  is  about  1/2500 
normal. 

(c)  TJjfelmann's  Test  for  Lactic  Acid. — In  each  of  3  test  tubes 
place  5  c.c.  of  1%  phenol  and  two  or  three  drops  of  ferric 
chloride  solution.  The  resulting  amethyst  liquid  is  Uifelmann's 
reagent.  To  one  tube  add  2  c.c.  dilute  lactic  acid.  To  the  second 
2  c.c.  0.2%  HCl  and  to  the  third  2  c.c.  acetic. 

(d)  To  a  small  amount  of  0.2%  HCl  add  Toepfer's  reagent 
(dimethyl-amino-azobenzene).  Make  slightly  alkaline  and  observe 
the  change  in  color. 

2.  Determination  of  Acidity  of  Gastric  Contents. — This  may 


266  PHYSIOLOGICAL    CHEMISTRY 

be  accomplished  as  follows :  The  subject  eats  only  a  cup  of  clear 
tea  and  two  pieces  of  dry  toast  for  breakfast.  One  and  one-half 
hours  after  breakfast,  his  stomach  contents  are  pumped  out  by 
means  of  a  stomach  pump.  Ten  c.c.  portions  of  this  liquid  are 
titrated  with  N/10  alkali  using  phenolphthalein  as  indicator. 
This  gives  total  acidity. 

Free  hydrochloric  acid  may  be  determined  by  titration  with 
N/10  alkali  using  Toepfer's  reagent  as  indicator,  or  by  the 
Gruenzberg  reagent  method  described  above.  The  gastric  eon- 
tents  may  be  tested  for  blood  by  the  guaiac  test  (as  performed 
in  the  study  of  hemoglobin). 

d.  Ferments.  The  gastric  juice  contains  two  or  perhaps 
three  ferments:  pepsin,  rennin  and  lipase. 

II.  Digestive  Action  of  Gastric  Juice. — 

a.  On  Proteins. — A  satisfactory  method  for  the  study  of  the 
digestive  action  of  gastric  ferments  on  proteins  is  the  use  of 
Mett's  tubes.  Mett's  method  consists  in  suspending  in  a 
digestive  mixture  short  glass  tubes  filled  with  coagulated  egg 
albumin.  After  incubating,  the  length  of  the  column  of  al- 
bumin which  has  been  digested  is  observed,  and  the  activity 
of  the  digestion  mixture  judged  accordingly. 

1.  Preparation  of  Mett's  Tiibes. — 

White  of  egg  is  beaten  to  break  the  reticulum,  strained 
through  linen  or  muslin,  and  allowed  to  stand  until  free  from 
air  bubbles.  The  egg  Avhite  is  then  drawn  up  into  lengths  of 
glass  tubing  having  an  inner  bore  of  1-3  mm.  The  tubing  is 
then  placed  on  a  wire  gauze  so  arranged  that  it  can  be  lowered 
into  the  inner  compartment  of  a  double  boiler.  The  water  in 
the  double  boiler  is  heated  until  that  in  the  inner  compart- 
ment reaches  a  temperature  of  85°  C.  The  gauze  is  then  low- 
ered into  the  water  and  allowed  to  remain  until  quite  cold. 
The  tubes  of  coagulated  albumin  may  be  preserved  by  covering 
the  ends  Avith  shellac.  For  use,  the  tubes  are  cut  into  2  cm. 
lengths,  breaking  the  tube  sharply  to  get  an  even  edge  of 
albumin. 


GASTRIC    DIGESTION  267 

2.  Arrange  six  test  tubes  as  follows,  numbering  them  Nos. 
1  to  6,  and  labelling  with  your  name. 

1.  5  c.c.  distilled  water. 

2.  5  c.c.  0.2%  hydrochloric  acid. 

3.  5  c.c.  water  -)-  10  drops  glycerine  extract  of 
pig's  stomach  prepared  above. 

4.  5  c.c.  0.2%  HCl  -f-  10  drops  glycerine  extract. 

5.  5  c.c.  0.2%  HCl  -f  10  drops  glycerine  extract, 
boil  thoroughly  3  or  4  minutes  and  cool. 

6.  5  c.c.  0.3%  sodium  carbonate  -j-  10  drops  glyc- 
erine extract. 

In  each  tube  suspend  a  Mett  tube  by  means  of  a  thread 
supported  by  a  match.  Make  sure  that  the  Mett  tube  hangs 
so  that  it  does  not  touch  the  bottom  of  the  test  tube.  The 
digestion  liquid  must  have  free  access  to  the  egg  white.  Place  in 
the  incubator  until  the  next  period.  After  a  minimum  time  of  24 
hours,  compare  the  amounts  of  digestion  in  the  different  tubes 
and  record  results.  A  slight  digestion  may  be  observed  in  No. 
2,  due  to  the  formation  of  acid  metaprotein.  Tube  No.  4  should 
show  good  digestion.  Tube  No.  6  may  show  slight  digestion 
due  to  formation  of  alkali  metaprotein.  Why  does  not  No.  3 
digest  ? 

b.  On  Milk. — The  ferment  rennin,  by  some  investigators  con- 
sidered to  be  identical  with  pepsin,  causes  the  clotting  of  milk 
due  to  precipitation  of  casein.  Calcium  salts  are  necessary  for 
this  process.  Prepare  two  test  tubes  each  containing  5  c.c.  of 
milk. 

1.  To  one  add  3  c.c.  ammonium  oxalate. 

2.  To  the  second  add  3  c.c.  distilled  water. 

As  the  rennin  in  the  glycerine  extract  is  very  likely  to  have 
become  inactive,  powder  a  rennin  tablet  and  add  half  of  the 
powder  to  each  tube. 

Place  the  tubes  in  water  at  40°  for  20  minutes.  No.  2  should 
clot.  No.  1  will  fail  to  do  so,  since  calcium  is  necessary  in  the 
clotting  process,  and  the  oxalate  will  have  precipitated  it  as  in- 
soluble calcium  oxalate.     To  the  oxalate  tube  add  3-4  drops  of 


268  PHYSIOLOGICAL    CHEMISTRY 

concentrated  calcium  chloride  solution.    The  milk  now  clots,  since 
calcium  is  supplied. 

c.  Gastric  juice  contains  also  a  lipase,  but  its  action  is  not 
extensive. 

d.  Products  of  Gastric  Digestion. — When  the  hydrochloric 
acid  stomach  mucosa  mixture  has  remained  in  the  incubator  for 
2  or  3  days  or  longer,  remove  it,  boil,  filter  and  neutralize  the 
whole  of  the  filtrate.  Any  metaprotein  will  precipitate.  Filter 
if  necessary  and  saturate  with  solid  ammonium  sulphate.  Pro- 
teoses precipitate.  Filter  and  test  for  peptones.  Gastric  diges- 
tion takes  the  proteins  no  lower  than  peptones. 

III.  Motor  Power  of  the  Stomach. — 

a.  Take  by  mouth  a  capsule  containing  0.1  gm.  iodoform.  This 
is  not  decomposed  until  it  reaches  the  intestine.  Test  saliva  for 
iodine  by  spitting  upon  starch  paper  and  dropping  one  drop  of 
cone,  nitric  acid  upon  the  spot.  A  positive  test  appears  in  from 
1  to  li'o  hours. 

IV.  Rate  of  Absorption  From  the  Stomach. — 

a.  Take  by  mouth  0.2  g.  potassium  iodine  in  a  capsule.  Test 
saliva  at  minute  intervals  for  iodine  as  in  III.  a.  A  positive  test 
appears  in  from  10  to  15  minutes. 

b.  Take  by  mouth  a  capsule  containing  1  gm.  of  salol.  In  the 
intestine  this  is  broken  up  into  phenol  and  salicylic  acid.  A 
violet  color  imparted  to  the  urine  by  a  few  drops  of  ferric 
chloride  indicates  salicylic  acid.  A  test  should  appear  in  from 
1  to  ll^^  hours. 


CHAPTER  IX 
PANCREATIC  DIGESTION.— BILE 

Pancreatic  Juice 

A  solution  of  pancreatic  ferments  may  be  prepared  by  ex- 
tracting a  pig's  pancreas  with  glycerine  or  with  water  contain- 
ing chloroform.  For  laboratory  purposes  it  is,  however,  more 
convenient  to  use  a  solution  of  commercial  pancreatic  powder. 

I.  Composition  of  Pancreatic  Juice.— 

a.  Natural  pancreatic  juice  contains  small  amounts  of  pro- 
teins and  other  organic,  substances. 

b.  Inorganic  salts,  chiefly  sodium  carbonate. 

c.  Ferments.     The  active  pancreatic  juice  contains  three 
important  enzymes :    tiypsin,  amylase,  and  lipase. 

II.  Digestive  Action. — 

a.  On  proteins. 

Prepare  four  test  tubes  as  follows : 

1.  f)  c.c.  neutral  pancreatic  solution. 

2.  5  c.c.  neutral  pancreatic  solution  -j-  2  drops  saturated  so- 
dium carbonate  solution.  This  gives  a  concentration  of  about 
0.2%  sodium  carbonate. 

3.  5  c.c.  neutral  pancreatic  solution  --}-  3  to  4  drops  of  10% 
HCl.    This  gives  a  concentration  of  about  0.2%  acid. 

4.  5  c.c.  neutral  pancreatic  solution  +  2  drops  saturated 
sodium  carbonate  solution,  boil  thoroughly  3  or  4  minutes  and 
cool. 

To  each  tube  add  a  Mett  tube  as  described  under  "  Gastric 
Digestion ' '  and  incubate  until  the  next  period. 
Examine  the  tubes  and  tabulate  results. 

Trypsin  acts  best  in  a  silghtly  alkaline  solution.     It  also  will 

269 


270  PHYSIOLOGICAL   CHEMISTKY 

act  in  neutal  or  even  faintly  acid  solution.    It  is,  of  course,  de- 
stroyed by  boiling. 

b.  On  Fats. — Pancreatic  juice  contains  a  lipase.  To  5  c.c.  milk 
add  ?>  or  4  drops  of  litmus  or  lacmoid  solution,  and  enough 
sodium  carbonate  to  produce  a  blue  color  (no  more).  Add  the 
amount  of  pancreatic  powder  which  can  be  taken  up  on  a  knife 
point.  Keep  at  body  temperature  for  some  time.  The  color 
turns  pink,  since  fats  are  split,  fatty  acids  set  free,  and  the  reac- 
tion becomes  acid.  If  the  test  is  allowed  to  stand  too  long,  an  acid 
reaction  will  result  from  the  souring  of  the  milk  (production  of 
lactic  acid  from  lactose).  The  bile  greatly  favors  the  digestion 
of  fats  by  pancreatic  juice. 

c.  On  Starch. — The  pancreatic  juice  contains  an  amylase, 
which  soon  becomes  inactive  in  artificial  preparations. 

d.  Products  of  Pancreatic  Digestion  of  Proteins. 

Recall  the  results  of  your  work  on  products  of  pancreatic 
digestion  under  amino  acids. 

Intestinal  Juice 

The  succus  entericus  or  intestinal  juice  plays  an  important  role 
in  digestion.  For  a  description  of  the  intestinal  ferments  and 
their  action,  the  student  is  referred  to  the  discussion  of  this  sub- 
ject in  the  text. 

BUe 

The  bile  is  secreted  by  the  liver  into  the  gall  bladder  and 
thence  delivered  to  the  intestine.  Bile  plays  an  important  part 
in  digestion.  Note  the  green  or  yellow  color.  Test  the  reaction 
with  litmus.    It  usually  is  neutral  or  slightly  alkaline. 

I.  Compasition. — 

a.  Inorganic  Material. — The  bile  contains  various  inorganic 
substances,  among  them  the  phosphates  of  calcium,  magnesium 
and  iron;  sodium  and  potassium,  both  in  the  form  of  chlorides 
and  combined  with  bile  acids  to  form  bile  salts;  sulphur  and 


PANCREATIC   DIGESTION  271 

phosphorus  in  organic  combination,  and  various  substances.    In- 
organic sulphates  are  absent  or  present  only  in  traces. 

b.  Bile  Acids  in  the  Form,  of  Alkali  Salts. 

1.  Pettenkofer's  test  for  hile  salts.  To  5  c.c.  of  bile  add  a  few 
grains  of  cane  sugar  or  a  few  drops  of  cane  sugar  solution.  With 
a  pipette  (caution:  do  not  draw  acid  into  the  mouth),  add  con- 
centrated H2SO4  to  form  a  layer  at  the  bottom,  or  allow  2-3  c.c. 
of  acid  to  run  down  the  side  of  the  tube.    Note  the  red  ring. 

2.  Extraction  of  hile  salts.  Bile  salts  may  be  prepared  by 
heating  bile,  charcoal  (enough  to  make  the  mixture  fairly  thick) 
and  5  volumes  of  alcohol  on  the  water  bath  for  20  minutes,  re- 
placing alcohol  lost  by  evaporation.  Filter  off  the  liquid  and  add 
ether  in  excess.    Bile  salts  are  precipitated. 

c.  Bile  Pigments. 

I.  Gmelin's  test.  To  about  5  c.c.  of  concentrated  HNO3  in  a 
test  tube,  add  2-3  c.c.  of  diluted  bile,  carefully,  so  that  the  two 
liquids  do  not  mix.    Note  the  colored  rings. 

d.  Mucin  and  Pseudomucin.  Bile  is  said,  by  some  authorities 
to  contain  mucin.  On  the  addition  of  5  volumes  of  alcohol  to 
bile  a  precipitate  is  formed.  This  is  called  pseudomucin,  and  is 
believed  by  some  to  be  a  phosphoprotein. 

e.  In  addition  to  the  above  constituents,  bile  also  contains 
small  amounts  of  fats,  lecithin,  phosphates,  cholesterol,  etc. 

II.  Effect  of  Bile  on  Surface  Tension. — Fill  one  small  beaker 
with  distilled  water.  To  a  second  beaker,  add  bile  diluted  with  4 
parts  of  water.  Sprinkle  a  few  grains  of  powdered  sulphur  over 
each.  In  the  beaker  containing  bile,  the  sulphur  sinks  to  the  bot- 
tom. The  surface  tension  of  the  water  has  been  reduced  so  that 
the  surface  film  is  no  longer  able  to  support  the  sulphur  grains. 

III.  Biliary  Calculi,  or  Gall  Stones,  are  of  four  kinds: 

a.  Those  made  up  of  calcium,  iron  or  copper  combined  with 

bile  pigments. 

b.  Cholesterin  calculi. 

c.  Talculi  of  calcium  phosphate  and  carbonate. 

d.  Calculi  of  combinations  of  the  above. 


272  PHYSIOLOGICAL    CHEMISTRY 

Analysis  of  gall  stones. — Extract  gall  stone  powder  with  a 
small  volume  of  ether.  Filter  through  a  dry  filter,  allow  ether 
to  evaporate  and  observe  the  residue  of  cholesterin  (1).  The  ma- 
terial which  did  not  dissolve  in  ether  consists  mainly  of  inorganic 
substances  and  bile  pigments.  Dissolve  out  the  inorganic  mia- 
terial  by  adding  a  small  amount  of  10%  HCl.  (See  2  a 
below.)  The  filtrate  contains  inorganic  material  (2)  and  the  resi- 
due consists  mainly  of  bile  pigments  (3). 

1.  (a)  Examine  under  a  microscope  the  crystals  left  on  evap- 
orating the  ether  extract.  If  the  crystals  are  not  well  formed,  dis- 
solve a  portion  of  them  in  a  very  small  quantity  of  alcohol  and 
allow  to  evaporate  slowly.  Draw  crystals.  They  should  be  large, 
colorless  plates. 

(b)  To  a  few  cholesterin  crystals  add  concentrated  H2SO4  and 
note  the  red  color. 

2.  Inorganic  Material. — Analyze  the  hydrochloric  acid  extract 
obtained  above  as  follows: 

(a)  Carbonates.  If  carbonates  were  present,  an  evolution  of 
CO;;  will  have  been  observed  on  the  addition  of  10%  HCl  above. 

(b)  PJiospJiates. — Evaporate  a  portion  of  the  HCl  extract  to 
dryness  on  the  water  bath,  take  up  with  a  few  drops  of  concen- 
trated HNO3,  dilute  with  a  few  cubic  centimeters  of  water  and 
add  ammonium  molybdate  in  excess.  Observe  the  yellow  crystals 
of  ammonium  phospho-molybdate. 

(c)  Calcium. — Evaporate  the  remainder  of  the  HCl  extract  to 
dryness,  take  up  with  a  few  cubic  centimeters  of  10%  acetic  acid 
and  add  ammonium  oxalate.    Calcium  oxalate  precipitates. 

3.  Bile  Pigments. — The  residue  insoluble  in  10%  HCl  is 
extracted  several  times  with  chloroform.  A  yellow  color  indicates 
bilirubin.  If  time  permits,  filter  the  chloroform  extract,  evapo- 
rate to  dryness  and  test  for  bile  pigments.  The  residue  insoluble 
in  chloroform  may  be  extracted  with  a  few  cubic  centimeters  of 
hot  alcohol,  filtered,  evaporated  and  the  resulting  residue  tested 
for  biliverdin. 


CHAPTER  XI 
URINE 

1.  Qualitative  Study 

For  the  qualitative  study  of  urine  each  student  will  need  2 
liters  of  urine.  (3  liters,  if  purine  bases  are  to  be  included.) 
The  urine  should  be  preserved  by  the  addition  of  a  5%  solution 
of  thymol  in  chloroform,  about  5  c.c.  per  liter  of  urine.  This 
preservative  should  be  placed  in  the  flask  before  the  urine  is  col- 
lected, as  otherwise  the  urine  may  decompose. 

1.  Inorganic  Constituents.— 

(a)  Chlorides. — Acidify  about  10  c.c.  of  urine  with  2-3  drops 
cone,  nitric  acid  and  add  a  drop  of  silver  nitrate.  If  chlorides 
are  present  in  normal  quantity,  a  solid  clump  of  silver  chloride 
will  sink  to  the  bottom.  If  but  small  amounts  of  chlorides  are 
present,  the  urine  becomes  only  cloudy.  If  an  attempt  be  made 
to  confirm  chlorides  by  dissolving  the  precipitate  in  ammonia,  a 
heavy  flocculent  precipitate  of  earthy  phosphate  will  be  thrown 
down.  Such  a  precipitate  might  be  filtered  off  however,  and  the 
dissolved  chlorides  reprecipitated  by  adding  nitric  acid. 

(b)  Phosphates. — There  are  two  general  classes  of  phosphates 
in  urine, — alkali,  i.e.,  sodium  and  potassium,  and  earthy,  i.e., 
calcium  and  magnesium.  These  phosphates  are  present  both  as 
mono-  and  di-hydrogen  salts,  e.g.,  NaaHPO^  and  NaHsPO^.  There 
also  is  phosphorus  in  urine  which  is  not  detected  in  the  usual 
precipitation  tests.  It  is  in  organic  combination  and  may  be 
detected  only*  after  fusion  with  an  oxidizing  agent. 

(1)  Earfhy  PhospJiates. — To  about  10  c.c.  urine,  add  ammonia 
to  alkalinity  and  warm.  A  flocculent  precipitate  is  earthy  phos- 
phates.    The  alkali  phosphates  remain  in  solution. 

(2)  Filter  off  the  precipitated  earthy  phosphates  and  add 

273 


274  PHYSIOLOGICAL    CHEMISTRY 

magnesia  mixture  to  the  filtrate.  Observe  a  precipitate  due  to 
alkali  phosphates.  The  fairly  soluble  alkali  phosphates  are  pre- 
cipitated as  magnesium  ammonium  phosphate. 

(c)  Sulphates. — Sulphur  is  present  in  three  forms:  In- 
organic sulphates,  ethereal  sulphates,  and  unoxidized  sulphur  in 
organic  combination.  To  determine  unoxidized  sulphur,  it  is 
necessary  first  to  fuse  with  an  oxidizing  agent. 

(1)  Inorganic  Sulphates. — To  about  10  c.c.  of  urine  add  2-3 
drops  of  concentrated  HCl  and  barium  chloride  in  excess.  The 
inorganic  sulphates  are  precipitated  as  barium  sulphate,  which 
will  settle  to  the  bottom  as  a  whitish  layer  on  standing. 

(2)  Ethereal  Sulphates. — Filter  off  the  precipitated  barium 
sulphate.  If  the  filtrate  does  not  come  through  clear,  mix  with 
it  a  small  amount  of  bismuth  subnitrate  and  filter  repeatedly 
through  the  same  filter.  The  bismuth  subnitrate  stops  the  larger 
pores  of  the  filter  paper,  thus  helping  to  keep  back  the  fine  par- 
ticles of  barium  sulphate.  To  the  clear  filtrate  add  about  2  c.c. 
cone.  HCl  and  boil  for  some  minutes.  Ethereal  sulphates  are 
split  up  in  this  process,  and  since  there  is  still  excess  of  barium 
chloride,  a  second  "crop"  of  barium  sulphate  is  obtained.  As 
the  amount  is  small,  it  may  not  be  seen  until  the  test  has  stood 
for  a  few  minutes,  when  the  precipitate  will  form  a  film  or  layer 
of  deposit  at  the  bottom  of  the  tube. 

(d)  Carbonates. — Evaporate  10  c.c.  urine  (Hood)  to  dryness 
on  the  water  bath.  Add  10%  HCl.  Observe  the  evolution  of 
carbon  dioxide  from  the  decomposed  carbonates. 

(e)  Ammonia. — To  about  10  c.c.  urine  in  a  test  tube,  add  so- 
dium carbonate  until  alkaline.  Moisten  a  piece  of  red  litmus 
paper  with  distilled  water  and  hang  in  the  mouth  of  the  tube. 
The  paper  will  be  turned  blue  by  the  ammonia  liberated. 

(f)  In  addition  to  the  above  inorganic  materials,  urine  con- 
tains vaying  amounts  of  sodium,  potassium,  calcium,  magne- 
sium, iron,  fluorine,  nitrates,  silicates,  traces  of  hydrogen  perox- 
ide, and  dissolved  gases,  e.  g.,  COo,  Ng,  and  Og,  besides  a  pos- 
sible number  of  casual  constituents  which  may  be  taken  in  the 
food  and  gotten  rid  of  by  way  of  the  kidneys. 


URINE 


275 


2.  Organic  Constituents. — 

(a)  Urea. 

(1)  Preparation  from  Urine. — Evaporate  500  c.c.  of  urine  on 
the  water  bath  to  the  consistency  of  a  thin  syrup.  Cool  and  add 
twice  the  volume  of  nitric  acid  (50%).  Allow  to  stand  in  a  cool 
place  over  night.  Filter  off  the  crystals  (dry  between  filter 
papers),  remove  crystals  from  the  filter,  and  dissolve  them  in  a 
small  amount  of  hot  water.  Add  a  little  potassium  permanga- 
nate solution  to  this  urea  nitrate  solution,  stirring  constantly, 
until  the  nitrate  solution  is  nearly  colorless.  Bring  the  solution 
to  a  boil,  and  add  first  solid  sodium  carbonate  and  then  solid 
barium  carbonate  until  COg  ceases  to  come  off,  and  the  solution 
is  neutral.  Evaporate  over  the  water  bath  to  dryness,  powder  the 
residue  and  extract  it  with  95%  warm  alcohol.  Filter  and  set  the 
filtrate  aside  to  cool.  Examine  the  crystals  under  the  micro- 
scope. 

(2)  Beaction  of  Urea. 

i.  Test  solubility  of  urea  in  alcohol. 

ii.  Heat  a  few  crystals  of  urea  gently  in  a  dry  test  tube. 
Add  a  small  volume  of  water  and  perform  the  biuret  test.  Since 
biuret  is  formed  when  urea  is  heated  dry,  a  positive  test  should 
result. 

iii.  Dissolve  a  few  crystals  of  urea  in  a  few  drops  of  water. 
Add  2  drops  of  cone.  HNOg.  Observe  the  crystals  of  urea  nitrate 
under  the  microscope,  and  draw.  Repeat  with  oxalic  acid  and 
observe  the  urea  oxalate. 

iv.  Dissolve  a  few  crystals  of  urea  in  a  few  cubic  centimeters 
of  water  and  add  sodium  hypobromite  solution. 

Note:    Sodium  hypobromite  is  prepared  by  adding  bro- 
mine water  to   sodium   hydrate   solution. 

Note  effervescence.  This  is  due  to  the  liberation  of  free  nitro- 
gen in  the  decomposition  of  urea  by  the  hypobromite. 

(b)  Uric  Acid. 

(1)  To  a  liter  of  urine  add  25  c.c.  cone,  hydrochloric  acid. 
Allow  to  stand  in  the  ice  chest  over  night.    Decant  carefully  from 


276  PHYSIOLOGICAL    CHEMISTRY 

the  reddish  brown  crystals  and  collect  them  on  a  small  filter. 
Examine  microscopically.  The  crystals  are  colored  dark  brown 
by  urine  pigment,  and  occur  in  a  variety  of  forms,  lozenge  or 
"gunboat"  shapes,  thick  rods,  etc.  These  crystals  are  not  pure 
but  serve  for  a  study  of  the  reactions  of  uric  acid. 

(2)  Reactions  of  uric  acid. 

1.  Solubility.  Test  solubility  of  uric  acid  in  ammonia,  so- 
dium hydroxide,  and  concentrated  sulphuric  acid.  Also  in  alco- 
hol, ether,  and  boiling  glycerol. 

ii.  Murexid  test.  To  a  few  crystals  of  uric  acid  in  an  evap- 
orating dish  add  2-3  drops  cone,  nitric  acid  and  evaporate  to 
dryness.  Cool  the  dish  and  add  a  drop  of  dilute  ammonia.  Ob- 
serve the  red  or  purple  spot.  This  test  is  given  also  by  xanthine. 
To  distinguish  between  the  two  substances  again  evaporate  to 
dryness.  The  color  disappears  if  the  substance  is  uric  acid.  If 
it  is  xanthine,  the  red  color  persists. 

iii.  Boil  a  few  crystals  of  uric  acid  with  a  small  amount  of 
Fehling  's  solution.  A  slight  reduction  will  occur,  but  it  may  be- 
come apparent  only  by  allowing  the  cuprous  oxide  to  settle  to 
the  bottom  of  the  tube.  This  should  occur  inside  of  ten  or  fif- 
teen minutes  after  the  boiling  (I/2  minute)  is  stopped.  This 
result  should  be  kept  in  mind  in  testing  supposed  diabetic  urines. 
The  amount  of  uric  acid  in  normal  urine  is  so  small,  however, 
that  it  will  not  reduce  Fehling 's  solution  preceptibly. 

(c)  Purine  Bases. 

(1)  Preparation  {Salkowski  Method). — To  a  liter  of  urine 
add  200  c.c.  of  magnesia  mixture.  Filter  off  the  precipitate,  add 
an  excess  of  strong  ammonia,  and  then  50-60  c.c.  of  3%  silver 
nitrate.  Allow  the  mixture  to  stand  for  an  hour  and  filter, 
washing  the  precipitate  to  remove  excess  of  silver.  Suspend  the 
precipitate  in  400  c.c.  boiling  water,  acidify  with  a  few  drops  of 
cone.  HCl.  Pass  hydrogen  sulphide  into  the  hot  solution  as  long 
as  a  precipitate  forms.  Boil  the  mixture  a  few  minutes  (Hood)  to 
remove  excess  H^S  and  filter  off  the  precipitated  silver  sulphide. 
Evaporate  the  clear  filtrate  to  dryness  on  the  water  bath.  The 
residue  contains  uric  acid  and  other  purine  bases.    Extract  the 


URINE  277 

residue  with  boiling  3%  sulphuric  acid.  This  dissolves  the  xan- 
thine bases  and  leaves  the  uric  acid  as  a  residue.  Allow  to  stand 
over  night,  filter,  and  make  the  filtrate  strongly  alkaline  with 
ammonia.  Repreeipitate  xanthine  bases  as  above  with  silver 
nitrate.  Filter,  suspend  the  precipitate  in  boiling  water  acid- 
ulated with  HCl.  Boil  vigorously  a  few  minutes  and  decom- 
pose the  silver  salts  with  HgS.    Filter,  and  evaporate  to  dryness. 

(2)  Reactions  of  XantMne  Bases. 

i.  Test  solubility  in  water,  alcohol,  acids  and  alkalies. 

ii.  Murexid  test.  Perform  this  test  as  described  under 
uric  acid.  Note  the  method  of  distinguishing  xanthine  from  uric 
acid. 

(d)  Creatinine. 

(1)  Preparation.- — Creatinine  may  be  prepared  from  urine  by 
precipitation  as  the  zinc  salt.  Its  reactions  may  be  studied, 
however,  without  isolation  from  the  urine. 

(2)  Reactions. 

i.  Weyl's  test.  To  about  10  c.c.  of  normal  urine  add  2-3 
drops  of  sodium  nitroprusside  and  then  2-3  drops  10%  sodium 
hydrate.  A  red  color  which  soon  fades  to  yellow  shows  the  pres- 
ence of  creatinine.  (Compare  with  Legal's  test  for  acetone.  See 
below.)  On  the  addition  of  glacial  acetic  acid  to  this  test,  a  pure 
solution  of  creatinine  gives  a  greenish  color,  which  serves  to  dis- 
tinguish it  from  acetone.  In  urine,  however,  this  reaction  with 
acetic  acid  is  not  easily  recognized. 

A  second  simple  way  to  determine  to  which  of  these  substances 
a  positive  test  is  due  is  to  distill  the  urine.  Acetone  will  pass 
over  in  the  first  few  cubic  centimeters  of  distillate,  whereas  crea- 
tinine will  not  distill. 

ii.  Jaffe's  test.  To  10  c.c.  of  urine,  add  about  1  c.c.  picric 
acid,  and  then  1-2  c.c.  of  10%  sodium  hydrate.  Observe  the  red 
color.  This  test  serves  as  the  basis  for  the  quantitative  estima- 
tion of  creatinine.     (See  below). 

(e)  Oxalic  Acid. — To  200  c.c.  urine  in  a  beaker  add  5  c.c. 
saturated  solution  of  calcium  chloride,  acidify  with  acetic  acid 


278  PHYSIOLOGICAL    CHEMISTRY 

and  set  aside  over  night.  Examine  the  sediment  under  the 
microscope  and  note  the  diagonally  crossed  crystals  of  calcium 
oxalate. 

(f)  Urinary  Indican.—Indican  is  usually,  but  not  always, 
found  in  the  urine.  It  is  formed  from  the  products  of  protein 
putrefaction  in  the  intestine.  To  15-20  c.c.  urine  in  a  test  tube 
add  2  c.c.  copper  sulphate  solution,  15  or  20  c.c.  cone.  HCl  and 
5  c.c.  chloroform.  Close  the  mouth  of  the  tube  with  the  thumb 
and  shake  cautiously  (over  the  sink).  Continue  the  shaking  for 
several  minutes.  The  ' '  indican ' '  is  oxidized  to  indigo,  which  dis- 
solves in  the  chloroform,  giving  it  a  blue  color.  The  depth  of 
color  developed  in  the  chloroform  indicates  roughly  the  amount 
of  indican  present. 

(g)  Pigments. — Urochrome  and  uroerythrin  are  the  most  im- 
portant urinary  pigments. 

(1)  TJrochrome  is  a  substance  which  gives  the  yellow  color  to 
urine. 

(2)  UroerytJirin.  This  red  pigment  gives  the  deep  reddish 
color  to  sediments  of  uric  acid  and  urates.  To  highly  colored 
urine  add  lead  acetate  solution,  and  allow  to  stand.  In  the  pres- 
ence of  uroerythrin  the  precipitate  will  be  a  beautiful  pink 
color. 

Uroerythrin  in  amounts  sufficient  to  give  a  strong  test  occurs 
somewhat  rarely;  in  case  your  specimen  gives  a  good  test,  hand 
the  test  tube  to  an  instructor  so  that  it  may  be  exhibited  to  the 
class. 

(h)  In  addition  to  the  above  constituents  the  urine  contains 
small  and  A^arying  amounts  of  various  substances,  e.g.,  hippuric 
acid,  allantoin,  aromatic  oxyacids,  compounds  containing  sulphur 
and  many  other  substances. 

2.  Collection  and  Preservation  of  a  Specimen  for  Quantitative 
Analysis — General  Properties 

As  the  composition  of  the  urine  voided  at  different  times  of 
the  day  varies  considerably,  it  is  customary  to  use  for  metabolism 
studies  a  complete  24-hour  sample.     This  may  be  obtained  as 


URINE  279 

follows :  On  rising  in  the  morning  empty  the  bladder  and  dis- 
card the  urine  voided.  Then  collect  all  urine  voided  during  the 
day,  and  the  first  voiding  on  rising  the  following  morning.  This 
constitutes  a  complete  24-hour  specimen.  For  the  observations 
in  this  section  and  the  quantitative  analysis  in  the  section  fol- 
lowing, a  complete  24-hour  specimen  should  be  used,  and  all 
determinations  made  on  the  same  specimen. 

As  some  urinary  constituents  decompose  on  standing,  it  is 
necessary  to  add  a  presei'vative  to  the  urine.  In  collecting  a  24 
hour  sample,  5  c.c.  of  5%  thymol  in.  chloroform  should  be  placed 
in  the  bottle  intended  for  collection  of  the  sample,  before  the 
urine  is  collected.  Neglect  of  this  precaution  may  lead  to  loss 
of  the  specimen  through  decomposition. 

a.  Volume.  Measure  in  a  large  cylinder  and  record  the  volume 
of  a  24-hour  sample. 

b.  Record  the  color,  odor,  transparency  and  presence  or 
absence  of  a  sediment. 

c.  Specific  gravity.  The  specific  gravity  may  be  determined 
either  gravimetrically  or  volumetrically.  For  most  clinical  work, 
the  determination  with  an  areometer  or  ' '  urinometer "  is  sufii- 
ciently  accurate. 

1.  Volumetrically. — Pour  the  urine  into  an  areometer  cylinder 
and  determine  the  specific  gravity  with  an  areometer  which  reg- 
isters 1.000  to  1.060.  Make  sure  that  the  instrument  floats  free 
in  the  urine  without  touching  the  walls  of  the  cylinder.  As  the 
instruments  are  standardized  for  a  definite  temperature  (usually 
15°  C.)  it  is  necessaiy  to  correct  for  any  variation  in  the  tem- 
perature of  the  urine  from  that  indicated  on  the  instrument. 
For  every  3°  C.  variation  a  correction  of  0.001  is  added  or  sub- 
tracted from  the  reading,  according  as  the  urine  is  warmer  or 
colder  than  the  standard  temperature.  Observe  the  temperature 
of  the  urine  and  make  any  necessary  correction  as  described 
above. 

2,  Gravimetrically. — If  a  gravimetric  determination  is  desired, 
it  may  be  made  as  follows :  Carefully  clean  and  dry  a  small 
weighing  bottle  and  weigh  quantitatively.     From  a  pipette  run 


280  PHYSIOLOGICAL    CHEMISTEY 

into  the  weighing  bottle  25  c.c.  of  distilled  water  at  room  tem- 
perature, stopper  the  bottle,  and  weigh. 

Note:  The  pipette  should  be  cleaned  with  cleaning  fluid 
and  then  thoroughly  rinsed,  first  with  tap  water  and  then 
once  or  twice  with  distilled  water.  If  the  liquid  gathers 
in  drops  on  the  inside  of  the  pipette  after  its  contents  have 
been  allowed  to  flow  out,  the  cleaning  should  be  repeated. 
On  emptying  a  perfectly  clean  pipette  or  burette,  the  in- 
strument should  be  so  clean  that  no  drops  remain  hanging 
on  the  inside  walls  of  the  instrument;  otherwise  the 
amount  delivered  will  not  correspond  to  the  amount  en- 
graved on  the  glass. 

In  filling  a  pipette,  draw  up  the  liquid  into  the  stem,  close  the 
top  with  the  index  finger,  and  allow  the  liquid  to  run  out  grad- 
ually, until  the  bottom  of  the  meniscus  is  level  with  the  gradua- 
tion on  the  stem. 

On  emptying  a  pipette,  allow  the  liquid  to  run  out  and  drain 
for  about  one-half  minute.  Then  touch  the  tip  of  the  pipette  to 
the  surface  of  the  liquid,  or  the  side  of  the  vessel.  Do  not  blow 
out  the  small  amount  of  liquid  remaining  in  the  tip.  This  is  the 
method  of  emptying  employed  in  the  standardization  of  accurate 
registered  pipettes.  In  the  case  of  accurate  work,  if  only  ordi- 
nary pipettes  or  burettes  are  available,  it  is  customary  to  stand- 
ardize such  instruments  by  weighing  the  amount  of  water  they 
will  deliver  and  calculating  the  volume. 

Clean  and  dry  the  weighing  bottle  and  measure  into  it  from 
the  same  pipette  25  c.c.  of  urine  and  weigh.  Tabulate  the 
weights  of  (a)  weighing  bottle,  (b)  weighing  bottle  +  water, 
(c)  weighing  bottle  -|-  urine.  Calculate  the  weights  of  water 
and  urine.  The  specific  gravity  of  the  urine  then  may  be  deter- 
mined by  dividing  the  weight  of  urine  by  the  weight  of  water. 
Since  equal  volumes  of  the  two  liquids  have  been  weighed,  it  is 
unnecessary  that  the  exact  volume  delivered  by  the  pipette  be 
known.  Weighing  the  amount  of  water  delivered  by  a  pipette 
or  burette  serves  to  determine  the  accuracy  of  such  an  instru- 
ment, for  a  given  weight  of  water  corresponds,  to  a  definite  vol- 
ume at  a  fixed  temperature.    In  standardizing  volumetric  appa- 


URINE  281 

ratiis  it  is  necessary  to  correct  for  temperature.  The  accuracy  of 
an  areometer  may  be  checked  by  trying  it  out  on  distilled  water, 
not  forgetting  the  correction  for  temperature. 

d.  Chemical  Reaction.  Test  the  chemical  reaction  of  the  urine 
with  litmus  paper.  A  24  hour  sample  is  usually  acid,  due  to  the 
presence  of  acid  phosphates  and  both  inorganic  and  organic 
acids.  If  the  urine  is  alkaline,  allow  the  litmus  paper  to  dry  on 
the  water  bath.  If  the  blue  color  disappears,  the  alkaline  reac- 
tion is  due  to  ammonia.    If  it  persists,  to  fixed  alkalies. 

e.  For  purposes  of  diagnosis  of  kidney  disorders,  it  is  some- 
times advisable  to  determine  both  the  freezing  point  and  the 
electrical  conductivity  of  the  urine.  The  technique  of  these  proc- 
esses, is,  however,  beyond  the  scope  of  this  course. 

3.  Quantitative  Analysis  (Make  all  Determinations  in 
Duplicate) 

1.  Total  Solids.^ — In  order  to  make  an  accurate  determination 

of  total  solids,  it  is  necessary  to  evaporate  a  measured  quantity 
of  urine  in  a  vacuum  at  room  temperature,  as  the  temperature  of 
the  boiling  water  bath  causes  decomposition  of  urea  and  loss  of 
ammonia  and  COg.  By  acidifying  slightly  with  acetic  acid  it  is 
possible  partially  to  avoid  this  source  of  error.  The  residue  is 
weighed  quantitatively,  and  the  total  solids  calculated  from  the 
result. 

An  approximate  but  usually  adequate  estimation  of  total  solids 
per  liter  may  be  calculated  by  using  Long's  coefficent,  2.6.  Multi- 
plying the  last  two  figures  (second  and  third  decimal)  of  the 
specific  gravity  at  25°  C.  by  2.6  gives  a  rough  idea  of  the  amount 
of  total  solids  per  liter. 

2.  Acidity.— For  the  determination  of  the  acidity  of  the 
urine,  and  for  various  others  of  the  methods  in  this  section,  acid 
or  alkali  of  standard  strength  is  required.  The  strength  most 
frequently  used  is  tenth  normal  (N/10).  A  normal  solution  of 
an  acid  is  of  such  strength  that  one  liter  of  the  acid  will  contain 
one  gram  equivalent  of  ionizable  hydrogen  (1.008  g.).  A  liter 
of  normal  hydrochloric  acid  thus  will  contain  a  weight  of  hydro- 


282  PHYSIOLOGICAL    CHEMISTRY 

chloric  acid  gas  equal  to  its  molecular  weight,  since  this  amount, 
36.46  g.,  will  contain  the  desired  amount  of  hydrogen.  If  sul- 
phuric acid  is  used,  the  amount  required  will  be  %  the  molec- 
ular weight,  since  the  formula  is  HgSO^,  and  if  this  weight  were 
taken,  it  would  contain  twice  as  much  hydrogen  as  required.  A 
normal  sodium  hydrate  solution  is  of  such  strength  that  it  will 
correspond  exactly  to  a  normal  acid  solution, — that  is,  it  will 
exactly  neutralize  an  equal  volume  of  normal  acid.  Since  one 
molecule  of  sodium  hydrate  will  neutralize  one  molecule  of 
hydrochloric  acid,  a  liter  of  normal  sodium  hydrate  must  con- 
tain the  same  number  of  molecules  as  a  liter  of  normal  hydro- 
chloric. This  result  will  be  obtained  if  the  solution  contains  an 
amount  equal  to  the  molecular  weight  of  sodium  hydroxide,  or  40 
grams,  since  40  grams  of  sodium  hydrate  will  neutralize  36.46 
grams  of  hydrochloric  acid.  If  barium  hydrate  were  used  Ba 
(OH) 2,  then  as  was  the  case  with  sulphuric  acid,  1/4  the  molec- 
ular weight  should  be  contained  in  a  liter  of  solution  if  it  is  to  be 
exactly  normal. 

From  the  amounts  required  for  a  normal  solution,  other 
strengths  such  as  N/10,  N/5,  etc.,  may  be  calculated. 

The  term  ' '  normal ' '  also  is  used  in  connection  with  oxidizing 
and  reducing  agents.  Thus  in  the  estimation  of  uric  acid  a 
N/20  potassium  permanganate  solution  is  used.  A  "normal" 
reducing  solution  must  yield  one  gram  equivalent  of  reducing 
hydrogen  per  liter,  and  a  normal  oxidizing  solution  must  furnish 
per  liter  enough  oxygen  to  oxidize  this  amount  of  hydrogen,  A 
normal  potassium  permanganate  solution  contains  a  weight  in 
grams  per  liter  equivalent  to  1/5  the  molecular  weight. 

(a)  Preparation  of  Standard  Acid  and  Alkali. — In  order 
to  prepare  an  acid  or  alkali  of  known  strength  it  is  necessary  to 
start  from  a  substance  of  known  composition  and  purity.  Var- 
ious methods  are  used  for  standardizing,  the  two  most  frequently 
used  being  the  oxalic  acid,  and  the  sodium  carbonate  methods. 

(1)  Oxalic  acid  metJiod.  Oxalic  acid  combines  with  varying 
amounts  of  water  of  crystallization.  By  drying  the  oxalic  acid  a 
day  or  two  in  a  desiccator  over  sulphuric  acid  of  specific  gravity 


URINE  283 

1.35,  however,  it  may  be  assumed  to  contain  2HoO.  Sixty-three 
gms.  of  the  crystals  so  prepared  are  dissloved  in  water  and  made 
up  of  1  liter.  This  solution  may  be  considered  normal  and  used  to 
standardize  an  unknown  solution  of  sodium  hydrate,  using 
phenolphthalein  as  indicator. 

(2)  Sodium  carbonate  method.  A  more  reliable  method  for 
preparing  a  standard  consists  in  heating  pure  sodium  bicarbonate 
in  a  platinum  dish.  The  dish  is  placed  in  an  air  bath  already 
heated  to  200°  C.  and  the  temperature  raised  to  270°-  280°  but 
not  above  300°.  It  is  heated  at  this  temperature  for  half  an 
hour,  then  cooled  in  a  desiccator,  and  before  the  sodium  carbon- 
ate is  quite  cool,  transferred  to  a  dry  stoppered  weighing  bottle. 

To  standardize  an  unknown  acid  solution,  rapidly  weigh  2-3 
gms.  of  carbonate  prepared  as  above,  dissolve  in  80-100  c.c.  of 
water,  add  2  drops  of  methyl  orange  and  titrate  with  the 
unknown  acid. 

An  exactly  normal  acid  should  neutralize  pure  sodium  car- 
bonate in  the  ratio  of  100  c.c.  acid  to  5.3  gms.  carbonate.  From 
the  results  of  the  titration,  which  should  be  done  in  duplicate, 
the  strength  of  the  unknown  acid  may  be  calculated,  and  the 
acid  diluted  accuately  to  the  required  strength.  After  dilution, 
the  acid  should  be  titrated  again  against  sodium  carbonate  to 
make  sure  that  the  dilution  was  accurate. 

After  making  an  accurate  solution  of  normal  acid  (H^SO^  or 
HCl)  a  solution  of  normal  sodium  hydrate  may  be  prepared, 
using  the  normal  acid  as  a  standard  and  titrating  with  alizarine 
red  or  methyl  red  as  indicator. 

If  time  does  not  permit  each  student  to  start  from  oxalic  acid 
or  sodium  carbonate  in  the  peparation  of  his  normal  solutions, 
standard  solutions  of  N/10  acid  and  alkali  prepared  as  above 
described  should  be  furnished  for  the  purpose  of  standardizing 
the  solutions  made  by  the  class. 

Note :  In  the  author 's  classes  it  has  been  customary  to 
furnish  to  the  class  N/IO  acid  and  alkali  for  the  purpose 
of  standardizing  solutions  prepared  by  them  as  described 
below,  thus  saving  much  time  for  the  class;  students  then 
use  their  own  N/10  solutions  in  their  work. 


284  PHYSIOLOGICAL    CHEMISTRY 

Each  student  should  prepare  one  standard  solution.  The  class 
may  be  divided  into  pairs,  one  student  of  each  pair  preparing  a 
standard  acid,  his  partner  a  standard  alkali.  The  solutions  pre- 
pared then  can  be  used  jointly  by  each  pair  of  students. 

(3)  Preparation  of  N/10  acid.  Bead  all  of  section  (3) 
before  beginning  your  work.  Cone,  hydrochloric  acid  is  about 
41%  by  volume,  that  is,  100  c.c.  contains  41  grams  of  hydro- 
chloric acid  gas.  Calculate  the  amount  of  cone,  acid  required  to 
make  2  liters,  1^^  or  1  liter  of  N/10  acid,  according  to  the  size  of 
the  large  glass  stoppered  bottle  with  which  you  are  provided,  and 
measure  this  amount  of  acid  into  your  large  bottle.  Add  dis- 
tilled water  to  make  the  volume  up  to  about  100  c.c,  less  than  the 
total  volume  for  which  you  have  made  your  calculation. 

With  a  clean  pipette  (see  note  under  Specific  Gravity)  meas- 
ure out  25  c.c.  of  the  acid  into  an  Erlenmeyer  flask.  The  pipette 
either  may  be  dry,  or  it  may  be  rinsed  out  once  with  the  solu- 
tion to  be  measured. 

Add  2-3  drops  of  alizarine  red  as  indicator,  and  from  a  burette 
(also  cleaned  as  above  and  either  dry  or  rinsed  with  N/10  NaOH) 
run  in  N/10  sodium  hydrate.  In  reading  the  burette,  read  the 
lowest  curve  of  the  meniscus.  Have  your  eye  on  a  level  with  the 
meniscus.  If  the  acid  were  exactly  N/10  it  would  require  exactly 
25  c.c.  of  the  alkali  to  neutralize  it.  Make  the  titration  in  dupli- 
cate. Duplicates  should  agree  within  0.1  c.c.  From  the  amount 
of  alkali  used  in  titration  calculate  the  amount  of  dilution  neces- 
sary to  make  the  acid  exactly  N/10. 

Example. — 25  c.c.  acid  used.  Amt.  N/10  alkali  to  neutralize 
the  acid  27.2  c.c.  Thus  25  c.c.  of  the  acid  used  contains  enough 
hydrochloric  acid  to  neutralize  27.2  of  N/10  alkali.  To  make  the 
acid  exactly  N/10  it  must  be  diluted  in  the  ratio  of  25 :  27.2. 

Measure  the  total  amount  of  your  acid  with  a  large  cylinder 
and  calculate  the  volume  of  water  necessary  to  dilute  it  to  tenth 
normal  acid.  Add  this  amount  of  water  from  a  cylinder,  and 
shake  thoroughly.  Allow  to  stand  for  a  few  minutes,  and  repeat 
the  titration  as  above. 

If  your  acid  does  not  check  with  the  N/10  alkali,  correct  it 


URINE  285 

again  as  above  by  the  addition  of  the  calculated  amount  of  water, 
or  of  the  calculated  amount  of  hydrochloric  acid  in  case  the 
solution  is  now  too  weak. 

Preserve  your  tenth  normal  solution  for  future  use. 

Rinse  out  the  pipette  and  burette  thoroughly. 

(4)  Preparation  of  a  standard  alkali.  Eead  over  carefully 
section  (3)  on  the  preparation  of  a  standard  acid,  as  the  prin- 
ciples involved  are  identical  with  those  for  the  preparation  of 
the  alkali. 

A  concentrated  solution  of  sodium  hydrate  will  be  used  as  a 
stock  solution.  The  strength  of  the  NaOH  will  be  found  on  the 
bottle.  Calculate  the  amount  required  to  make  up  2,  ly^,  or  1 
liter  of  N/10  alkali  according  to  the  size  of  the  glass  stoppered 
bottle  in  your  desk. 

Measure  this  amount  into  the  large  bottle  and  add  enough  dis- 
tilled water  to  make  the  volume  up  to  about  100  c.c.  less  than 
the  total  volume  for  which  you  have  made  your  calculation. 

In  titration  with  alizarine  red  as  an  indicator  it  is  necessary  to 
run  alkali  into  acid.  Accordingly,  measure  25  c.c.  of  the  standard 
N/10  acid  furnished  into  a  clean  Erlenmeyer  by  means  of  a  clean 
pipette  (see  note  under  Specific  Gravity).  Add  2-3  drops  of 
alizarine  red. 

Clean  your  burette  and  either  dry  it  or  rinse  with  the  solution 
to  be  used  in  it.  "Where  the  supply  of  liquid  is  adequate,  the 
second  method  is  easier. 

Fill  the  burette  with  your  alkali,  and  titrate  the  standard  acid. 
Make  a  duplicate  titration  to  check  your  result.  The  duplicate 
titrations  should  agree  within  0.1  c.c. 

If  your  alkali  were  exactly  N/]0,  it  would  require  25.0  c.c.  to 
neutralize  the  25  c.c.  acid.  As  you  have  made  it  up  slightly 
stronger  than  this,  it  will  require  somewhat  less  than  25  c.c. 
alkali  to  neutralize  the  25  c.c.  of  acid. 

From  the  amount  of  alkali  used  calculate  the  amount  of 
dilution  necessary  to  make  the  alkali  exactly  N/10. 

Example:  25  c.c.  N/10  acid  is  neutralized  by  23.2  c.c.  alkali. 

Thus  23.2  c.c.  of  alkali  contains  enough  NaOH  to  neutralize 


286  PHYSIOLOGICAL    CHEMISTEY 

25  c.c.  N/10  acid.  To  make  the  alkali  exactly  N/10  it  should 
be  diluted  in  the  ratio  of  23.2 :25. 

Measure  the  total  volume  of  your  alkali  in  a  large  cylinder 
(first  returning  any  of  the  solution  remaining  in  the  burette) 
and  calculate  the  amount  of  water  necessary  to  dilute  it  to 
tenth  normal  alkali.  Add  this  amount  of  water  from  a  cylinder, 
shake  well  and  allow  to  stand  for  a  few  minutes. 

Rinse  out  the  burette  with  the  diluted  alkali,  and  repeat  the 
titration  against  standard  acid. 

If  your  alkali  does  not  check  with  the  standard  acid,  correct 
it  again  by  adding  the  calculated  amount  of  water,  or  the  cal- 
culated amount  of  sodium  hydrate  if  the  solution  has  been 
made  too  dilute. 

Preserve  your  tenth  normal  solution  for  future  use. 

(b)  Determination  of  Acidity  of  Urine. 

(1)  Preliminary  Experiments. 

i.  Measure  out  10  c.c.  of  potassium  dihydrogen  phosphate 
into  each  of  3  small  beakers  or  Erlenmeyer  flasks,  and  label  1, 
2  and  3. 

To  No.  2  add  3-4  drops  sat.  CaCla  soln. 

To  No.  3  add  3-4  drops  sat.  CaCL  sol.  +  about  10  c.c.  15% 
neutralized  potassium  oxalate  solution.  Titrate  all  3  solutions 
with  N/10  NaOH,  using  phenolphthalein  as  indicator.  Observe 
that  the  presence  of  calcium  increases  the  titration  figure.  The 
phosphates  of  the  alkaline  earths  precipitate  in  alkaline  solu- 
tion. On  adding  oxalate,  however,  the  disturbing  influence  of 
calcium  is  nullified,  since  it  is  precipitated  as  calcium  oxalate. 

(2)  Acidity  of  Urine. — Folin's  Method. 

i.  Measure  25  c.c.  of  urine  into  an  Erlenmeyer  flask.  Add 
1-2  drops  phenolphthalein  and  10  c.c.  neut.  potassium  oxalate 
solution.  Shake  vigorously  for  1-2  minutes  and  titrate  at  once 
with  N/10  NaOH  to  the  first  faint  but  permanent  pink.  From 
the  value  obtained  calculate  the  total  acidity  of  the  24  hour 
specimen  in  cubic  centimeters  of  normal  acid. 

ii.  Repeat  the  process  described  in  i,   omitting  the  addition 


URINE  287 

of  oxalate.     Consider  the  result  in  connection  with  your  ob- 
servations in  the  preliminay  experiments  under  section  (1). 

3.  Total  Nitrogen.    (Kjeldahl  Method).— 

This  method  is  of  utmost  importance  and  is  widely  used  for 
estimating  total  nitrogen.  The  various  nitrogen  compounds 
are  broken  down  by  heating  with  concentrated  sulphuric  acid, 
the  nitrogen  being  converted  into  ammonia,  and  the  carbon  into 
carbon  dioxide.  The  ammonia  is  retained  in  the  solution  as 
(NH4)2S04.  It  then  is  liberated  by  the  addition  of  sodium 
hydrate  and  distilled  into  a  known  volume  of  N/10  hydrochloric 
or  sulphuric  acid.  The  excess  of  N/IO  acid  is  then  determined 
by  titration  and  the  amount  of  ammonia  calculated. 

In  analyzing  liquids  it  is  customary  to  use  5  or  10  c.c,  ac- 
cording to  the  nitrogen  content.  If  the  material  is  a  solid,  1 
gram  accurately  weighed  is  the  usual  amount. 

With  a  pipette  measure  5  c.c.  urine  into  a  500  c.c.  Kjeldahl 
flask.  Add  8-10  g.  potassium  sulphate  which  raises  the  boiling 
point,  15  c.c.  of  concentrated  HgSO^  and  2  c.c.  of  5%  copper 
sulphate  which  acts  as  a  catalyser. 

Heat  the  flask  in  an  inclined  position  over  a  small  flame  until 
the  contents  become  clear  and  pale  green.  There  must  be  no 
suggestion  of  yellow  and  no  black  specks  of  unoxidized  carbon 
anywhere  in  the  liquid  or  on  the  inner  surface  of  the  flask.  If 
black  specks  are  present,  remove  the  flask  from  the  flame  and 
by  careful  shaking,  rinse  them  down.  Do  not  attempt  to  rinse 
them  down  by  adding  water. 

Note :  Injurious  fumes  are  given  off  during  the  heating 
in  the  Kjeldahl  analj^sis,  so  that  it  should  never  be  carried 
out  in  the  open  laboratory,  but  in  a  well  drawing  hood,  or 
other  arrangement  for  carrying  off  the  fumes. 

When  digestion  is  complete,  allow  the  liquid  to  cool  and 
about  half  fill  the  flask  with  distilled  water.  The  nitrogen  now 
is  in  solution  in  the  form  of  ammonium  sulphate.  It  must  be 
set  free  as  ammonia  and  driven  over  by  distillation  into  a 
known  volume  of  standard  acid. 


288 


PHYSIOLOGICAL    CHEMISTRY 


Arrange  apparatus    (condenser,   connections,   etc.)    to   distill 
off  the  ammonia.     The  most  satisfactory  receiving  flask  is  a 


Fig.  3. — Apparatus  for  Distilling  Total  Nitrogen  by  Kjeldahl  Method. 


Woulff  bottle.     Connect  the  delivery  end  of  the  condenser  with 
one  neck  of  the  Woulff  bottle  by  means  of  rubber  and  glass 


URINE  289 

tubing,  and  a  stopper.  In  the  other  neck  of  the  Woulff  bottle 
fix  a  cork  through  which  passes  the  narrow  tube  of  a  CaClg 
tube.    Charge  the  bulb  of  the  CaCL  tube  with  glass  pearls. 

Prepare  the  Woulff  bottle  by  running  into  it  over  the  glass 
pearls  in  the  calcium  chloride  tube,  50  c.c.  N/10  hydrochloric 
acid  measured  with  a  pipette,  add  2-3  drops  alizarine  red  and 
connect  the  bottle  by  the  other  neck  with  the  delivery  tube  of 
the  distilling  apparatus.  Set  the  digestion  flask  on  a  ring  over 
the  flame  and  make  sure  that  all  connections  fit  properly.  Add 
a  small  quantity  of  talcum  powder  to  prevent  irregular  boiling 
(bumping)  but  not  more  than  the  amount  that  can  be  taken  up 
on  the  tip  of  a  knife  blade.  Add  a  drop  of  alizarine  and  then 
about  50  c.c.  of  sat.  NaOH  to  neutralize  the  sulphuric  acid. 
In  adding  the  alkali,  the  flask  should  be  tilted  somewhat  and 
the  sodium  hydrate  poured  down  the  side  of  the  tube  so  that 
it  will  form  a  layer  underneath  the  acid.  This  is  to  avoid 
possible  loss  of  ammonia.  Stopper  the  flask  immediately,  make 
sure  that  the  stopper  fits  tightly,  and  rotate  the  flask  gently 
so  as  to  mix  the  contents  thoroughly.  If  the  liquid  does  not 
turn  pink  or  purple,  add  more  alkali  as  above.  Distill  the  lib- 
erated ammonia  into  the  standard  acid.  When  about  %  of 
the  water  has  distilled  over,  remove  the  Woulff  bottle  and  test 
the  next  drop  of  distillate  for  ammonia  by  letting  it  fall  into  a 
small  beaker  containing  distilled  water  and  a  drop  of  alizarine. 
If  the  indicator  turns  pink,  replace  the  Woulff  bottle  and  con- 
tinue the  distillation  until  the  liquid  coming  over  no  longer 
contains  ammonia.  When  the  ammonia  is  all  over,  discontinue 
the  distillation,  rinse  down  the  glass  pearls  with  distilled 
water,  and  titrate  the  excess  of  N/10  HCl  with  standard  alkali. 

Subtracting  the  amount  of  alkali  used  from  the  total  acid 
(50  c.c.)  gives  the  volume  of  acid  neutralized  by  the  ammonia. 

Multiply  this  figure  by  the  weight  of  nitrogen  in  1  c.c.  of 
N/10  ammonia  (1.401  mg.).  This  gives  the  weight  of  nitrogen 
in  5  c.c.  urine.  Calculate  the  weight  of  nitrogen  in  the  24  hour 
sample. 


290  PHYSIOLOGICAL    CHEMISTRY 

In  accurate  work,  a  blank  determination  should  be  made  to 
estimate  the  amount  of  nitrogen  in  the  reagents  used, 

4.    Ammonia. — 

(a)  Folin  Air  Current  BletJiod.—The  most  satisfactory 
method  for  determining  ammonia  in  urine  is  that  of  Folin. 
This  consists  in  measuring  a  given  volume  of  urine  into  a  tall 
cylinder  (B)  fitted  with  a  two  hole  rubber  stopper.  Through 
one  hole  passes  a  glass  tube  reaching  to  the  bottom  of  the 
cylinder  for  the  admission  of  compressed  air.  Through  the 
second  hole  a  glass  tube  leads  into  a  receiving  flask  (C)  pro- 
vided with  a  Folin  absorption  tube.  In  this  flask  is  placed  a 
measured  volume  of  N/10  acid,  and  enough  distilled  water  to 
cover  the  holes  in  the  Folin  absorption  tube.  The  ammonia  is 
liberated  from  the  urine  by  adding  solid  sodium  carbonate. 

Into  the  receiving  flask  measure  20  c.c.  N/10  acid  and  add 
two  drops  of  alizarine  red.  Add  enough  distilled  water  to 
well  cover  the  bell  of  the  Folin  absorption  tube.  Into  the  tall 
cylinder  measure  25  c.c.  of  urine.  Cover  with  a  thin  layer  of 
kerosene  to  prevent  foaming.  Make  sure  that  all  connections 
are  tight  and  everything  in  readiness  to  start  the  air  current. 
The  compressed  air  should  be  run  through  10%  HgSO^  to 
remove  possible  ammonia.  Add  about  1  gm.  of  anhydrous 
sodium  carbonate  to  the  urine  and  stopper  the  cylinder  at  once. 
Pass  a  fairly  rapid  stream  of  air  through  the  apparatus,  being 
careful  not  to  blow  the  contents  of  the  cylinder  into  the  acid 
bottle.  A  loose  plug  of  cotton  may  be  introduced  in  the  path 
of  the  air  passing  from  urine  to  acid  by  including  a  calcium 
chloride  tube  in  the  circuit.  This  will  prevent  the  blowing 
over  of  any  particles  of  carbonate.  Eun  the  air  current  for 
11/2  hours.  Disconnect  the  acid  container,  rinse  and  remove  the 
Folin  absorption  tube,  and  titrate  the  remaining  acid  with  N/10 
alkali. 

Subtract  the  volume  of  alkali  used  in  titration  from  the 
original  volume  of  N/10  acid.  The  result  is  the  amount  of  N/10 
acid  neutralized  by  the  ammonia.    One  cubic  centimeter  of  N/10 


URINE 


291 


acid  corresponds  to  .0017  grams  of  NH3  or  .0014  grams  of  N. 
Calculate  the  amount  of  ammonia  nitrogen  in  the  24  hour 
specimen  and  also  the  per  cent  of  the  total  nitrogen  present 
as  ammonia. 


Fig.  4. — Folin's  Apparatus  roR  Ivsti.mating  Ammonia. 

A.  Wash  bottle  containing  acid. 

B.  Tall   aerometer   cylinder   containing   urine. 

C.  Bottle  containing  standard  acid. 

D.  Calcium    chloride    tube,    loosely    packed    with    cotton    wool,    to    prevent    any    sodiur 

carbonate  being  carried  over   into   C. 

E.  Folin's  absorption  tube,  to  bring  the  air  into  intimate  contact  with  the  acid. 


292  PHYSIOLOGICAL   CHEMISTRY 

(b)  Clinical  Method.  Shijf-Malfatti. — This  method  depends 
on  the  fact  that  when  neutral  solutions  of  ammonium  salts  are 
treated  Avith  formaldehyde,  urotr opine  (hexamethylene  tetra- 
mine)  is  formed  and  a  definite  amount  of  free  acid  is  liberated. 

6  HCHO  +  2  NHJ^SO,  =N,(CH,)«  +  2  H,SO,  +  6HoO. 

This  method  is  not  accurate,  but  serves  for  most  clinical  pur- 
poses. 

Measure  25  c.c.  urine  into  a  flask  and  dilute  with  5  volumes 
of  water.  Add  4  or  5  drops  of  phenolphthalein  and  5  c.c.  of  a 
saturated  solution  of  potassium  oxalate.  Titrate  with  N/10 
NaOH  to  a  faint  permanent  pink. 

The  formalin  solution  is  prepared  by  adding  3  volumes  of 
water,  a  few  drops  of  phenolphthalein,  and  titratang  with 
N/10  NaOH  to  a  faint  pink  to  neutralize  the  acid  present  in 
the  formalin  solution.  Add  30-40  c.c.  of  neutralized  formalin 
to  the  neutralized  urine.  The  color  disappears,  for  free  acid 
is  set  free  as  is  shown  in  the  above  equation.  Titrate  again 
to  a  faint  permanent  pink.  The  amount  of  alkali  corresponds 
to  the  amount  of  decinormal  ammonia  and  ammonium  salts 
present  in  the  urine.  Calculate  the  amount  of  ammonia 
N  in  25  c.c.  of  urine,  and  from  this  the  amount  in  your  24-hour 
specimen. 

Calculate  the  per  cent  of  the  total  nitrogen  which  is  present 
as  ammonia  nitrogen, 

5.  Urea. — 

Plimmer  and  Skelton  Modification  of  the  Marshall  Urease 
Method. — This  method  consists  in  decomposing  the  urea  by 
means  of  the  enzyme  urease,  which  is  found  in  the  soy  bean. 
The  nitrogen  is  converted  into  ammonia  and  the  ammonia  esti- 
mated. Of  the  various  recent  modifications  of  the  urease 
method  this  is  perhaps  the  best  suited  for  use  in  a  large  class 
unless  exceptional  laboratory  facilities  are  available. 

The  apparatus  required  is  identical  with  that  described  in  the 
Folin  ammonia  determination  above. 


URINE  293 

Measure  about  50-60  c.e.  of  distilled  water  into  the  tall  cylin- 
der. Add  1  gm.  of  finely  powdered  soy  bean,  5  c.e.  of  urine 
from  a  pipette,  and  kerosene  or  liquid  paraffin  to  prevent 
foaming.  Connect  with  a  receiving  bottle  containing  50 
c.e.  of  tenth  normal  acid  accurately  measured  and  two 
drops  of  alizarine  red.  Stand  the  cylinder  in  vessels  con- 
taining water  at  35°-40°  C.  The  w^ater  in  the  outside  vessel 
should  be  kept  at  this  temperature  by  the  addition  of  hot 
water  as  required.  Run  an  air  current  through  the  series  to 
drive  the  liberated  ammonia  into  the  acid  bottle  where  it  will 
be  absorbed.  As  the  air  from  the  compressed  air  tap  may  con- 
tain traces  of  ammonia  it  should  be  ran  through  10%  sulphuric 
acid  washing  bottles  before  being  run  into  the  urine  cylinder. 
After  about  an  hour,  disconnect  the  cylinder  and  add  about  a 
gram  of  anhydrous  sodium  carbonate  to  the  urine.  Connect 
again  and  run  the  air  current  for  another  hour.  Titrate  the 
excess  of  acid  in  the  acid  bottle  with  N/10  NaOH.  Subtracting 
this  value  from  the  total  amount  of  acid  used  will  give  the 
amount  of  acid  neutralized  by  the  liberated  ammonia.  Calcu- 
late the  weight  of  nitrogen  liberated  from  the  volume  of  urine 
used  and  from  this,  the'  amount  present  in  the  total  24-hour 
specimen.  This  value  represents  the  amount  of  nitrogen  pres- 
ent as  urea  -\-  ammonia.  Subtract  the  value  found  above  for 
ammonia.  The  result  will  be  the  amount  of  nitrogen  present 
as  urea.  Calculate  the  per  cent  of  the  total  nitrogen  which  is 
present  as  urea. 

6.  Uric  Acid. — 

Folm-Scliaffer  MetJiod. — Phosphates  and  some  organic  ma- 
terial are  first  precipitated  and  filtered  off.  The  uric  acid  is 
then  precipitated  as  ammonium  urate,  and  titrated  with  N/10 
potassium  permanganate. 

To  200  c.e.  of  urine  in  a  beaker,  add  50  c.e.  of  Folin-Schaf- 
fer  reagent  (50%  ammonium  sulphate,  0.5%  uranium  acetate 
and  0.5%  acetic  acid).  Allow  to  stand  for  Yo  hour  and  filter 
through  a  dry  filter.  Measure  out  125  c.e.  of  the  filtrate  (cor- 
responding to  100  c.e.  urine),  add  about  5  c.e.  of  ammonia  and 


294  PHYSIOLOGICAL    CHEMISTRY 

let  stand  for  48  hours.  Filter  cautiously  through  a  hard  filter, 
using  a  glass  rod  to  avoid  loss  of  liquid.  Rinse  out  the  beaker 
several  times  with  10%  ammonium  sulphate  solution,  each  time 
pouring  the  rinsing  liquid  onto  the  filter  after  it  has  drained 
completely,  thus  rinsing  both  beaker  and  precipitate  with  the 
same  liquid.     This  rinsing  should  be  repeated  5  or  6  times. 

Carefully  remove  the  filter  paper  from  the  funnel,  open  it 
and  rinse  the  precipitate  into  the  beaker  from  which  it  was 
just  removed,  taking  care  that  all  the  crystals  are  transferred 
to  the  beaker.  It  is  most  satisfactory  to  use  hot  water  for  the 
rinsing.  No  color  should  remain  on  the  filter  paper.  {Note. — 
Do  not  heat  the  water  in  a  thick  glass  wash  bottle.  Heat  in  a 
beaker  and  pour  into  the  wash  bottle).  The  total  volume  of  the 
rinsings  should  be  as  close  as  possible  to  100  c.c.  Cool  to  room 
temperature. 

Add  15  c.c.  of  cone.  H^SC^  and  titrate  the  hot  liquid  at  once 
with  N/20  potassium  permanganate.  The  liquid  should  be 
shaken  or  stirred  ■  continuously.  At  first  the  color  of  the  per- 
manganate will  disappear  instantly  without  diffusing  through 
the  liquid.  The  first  instantaneous  pink  color  throughout  the 
entire  liquid  is  taken  as  the  end  point  of  the  titration.  This 
color  will  disappear  very  quickly,  but  the  addition  of  another 
drop  also  will  cause  a  pink  color  to  diffuse  for  a  very  brief  time 
throughout  the  entire  liquid.  Each  c.c.  of  N/20  permanganate 
=  3.76  mg.  uric  acid.  Calculate  the  amount  of  uric  acid  in  the 
volume  of  urine  used  (100  c.c).  From  this  calculate  the 
amount  of  uric  acid  in  your  24  hour  specimen. 

From  the  formula  of  uric  acid  calculate  the  percentage  of 
nitrogen  it  contains.  From  this  and  the  results  obtained  above, 
calculate  the  amount  of  nitrogen  in  the  total  uric  acid.  Calcu- 
late the  per  cent  of  the  total  nitrogen  present  as  uric  acid  nitro- 
gen, 

7.  Hippuric  Acid. — 

Hippuric  acid  may  be  estimated  by  the  method  of  Folin  and 
Flanders  which  consists  in  hydrolyzing  the  hippuric  acid  and 
estimating  the  benzoic  acid  liberated. 


URINE  295 

8,  Purine  Bases. — 

Purine  bases  may  be  estimated  by  a  method  similar  in  prin- 
ciple to  the  Salkowski  Method  for  isolating  these  compounds. 
For  details  consult  a  larger  work. 

9.  Creatinine  (Folin.) — 

The  estimation  of  creatinine  is  a  colorimetric  method,  based 
on  the  Jaffe  reaction  already  familiar  to  the  student.  Picric 
acid  is  reduced  to  picramic  acid  and  the  red  color  produced  by 
the  latter  in  alkaline  solution  is  compared  with  a  N/2  potas- 
sium bichromate  solution  in  a  Duboscq  colorimeter. 

Measure  10  c.c.  urine  into  a  500  c.c.  volumetric  flask  ,add  15 
c.c.  of  a  saturated  solution  of  picric  acid,  and  5  c.c.  10%  NaOH. 
Shake  well  and  allow  the  mixture  to  stand  5  minutes.  Employ 
this  interval  to  become  familiar  with  the  colorimeter. 

Before  putting  any  liquid  into  the  cups  put  them  in  place 
and  run  both  prisms  down  until  they  touch  the  bottoms  of  the 
cylinders.  Read  both  sides  of  the  instrument.  Both  readings 
should  be  0.0.  If  they  are  not,  record  the  readings  carefully 
and  take  them  into  consideration  in  making  readings  during  the 
analysis.  Be  careful  about  spilling  liquid  upon  the  instrument. 
In  case  any  liquid  is  spilled  upon  the  instrument,  wipe  it  off 
carefully. 

Pour  a  little  N/2  potassium  bichromate  into  each  of  the 
cylinders  of  the  Duboscq  colorimeter.  Adjust  the  depth  of  the 
solution  in  the  left  hand  cylinder  exactly  to  the  8  mm.  mark. 
(If  the  reading  on  this  side  was  not  0.0  when  the  prism  was  run 
to  the  bottom  of  the  cup,  make  the  necessaiy  correction  so  that 
the  distance  between  the  prism  and  the  bottom  of  the  cylinder 
is  exactly  8.0  mm.)  Practice  matching  the  color  in  the  two 
halves  of  the  field  in  order  to  insure  greater  accuracy  in  the 
examination  of  the  unknown.  Since  the  two  bichromate  solu- 
tions are  of  equal  strength,  the  readings  should  be  identical,  no 
two  readings  differing  by  more  than  0.1  to  0.2  mm.  from  the 
true  value  (8  mm.).  Always  make  four  readings.  Disregard 
the  first,  which  is  apt  to  be  inaccurate,  and  take  the  average  of 


296  PHYSIOLOGICAL    CHEMISTRY 

the  remaining  three  provided  they  do  not  vary  more  than  0.2  mm. 

Exactly  at  the  close  of  the  5  minutes,  dilute  the  unknoM^n 
solution  to  the  500  c.c.  mark  shaking  the  flask  during  the 
addition  of  the  water  to  insure  complete  mixing.  Stopper  care- 
fully and  thoroughly  mix  the  solution.  Pour  out  the  bichro- 
mate from  the  right  hand  cylinder,  rinse  carefully  first  with 
water  and  then  with  the  solution  to  be  analyzed,  and  fill  the 
cylinder  half  full  of  the  liquid.  Match  the  color  by  moving  the 
prism  in  the  unknown  solution,  making  three  or  four  rapid 
readings. 

Ordinarily  10  c.c.  of  urine  is  used  for  this  determination,  but 
if  the  content  of  creatinine  is  above  15  mg.  or  below  5  mg.  the 
determination  should  be  repeated  with  a  volume  of  urine  con- 
taining an  amount  of  creatinine  between  5  and  15  mg.  This  is 
an  essential  point,  as  the  method  is  accurate  only  within  these 
limits. 

By  experiment  it  has  been  determined  that  10  mg.  of  pure 
creatinine  gives  a  color  of  such  strength  that  8.1  mm.  of  such  a 
solution  exactly  matches  8.0  mm.  of  bichromate  under  the 
above  conditions.  From  the  reading  obtained  with  the  unknown 
solution  it  thus  is  possible  to  calculate  the  amount  of  creatinine 
present  in  the  volume  of  urine  used.  Eemember  that  the  larger 
the  amount  of  creatinine,  the  deeper  will  be  the  color,  and  the 
shorter  the  column  of  liquid  necessary  to  match  the  color  of 
the  standard.  If  a  reading  of  8.1  mm.  corresponds  to  a  creat- 
inine content  of  10  mg.,  calculate  the  amount  of  creatinine  cor- 
responding to  the  reading  of  your  solution,  not  forgetting  that 
the  proportion  will  be  inverse.  Calculate  the  amount  of  creat- 
inine in  the  volume  of  urine  used  and  in  the  24-hour  specimen. 

From  the  formula  of  creatinin  calculate  what  per  cent  of 
nitrogen  it  contains,  and  then  the  amount  of  creatinine  nitro- 
gen present  in  the  24-hour  sample.  Calculate  the  per  cent  of 
the  total  nitrogen  which  this  represents. 

10.  Indican. — 

By  applying  the  qualitative  test  for  indican,  already  per- 
formed by  the  student,  and  comparing  the  color  produced  with 


URINE 


297 


that  of  Fehling's  solution  as  a  standard,  at  least  a  comparative, 
if  not  a  quantitative  estimation  of  indiean  may  be  made. 

11.  AUantoin. — 

For  details  of  the  estimation  of  allantoin  consult  a  larger 

work. 

12.  Oxalic  Acid. — 

Oxalic  acid  may  be  estimated  by  the  isolation  of  its  calcium 
salt. 

13.  Chlorides.— 

Yolliard  Metliod. — All  of  the  chlorides  of  the  urine  are  pre- 
cipitated by  adding  excess  (a  known  volume)  of  standard  sil- 
ver nitrate.  The  excess  silver  nitrate  then  is  determined  by 
titration  with  thiocyanate,  using  a  ferric  salt  as  indicator.  The 
thiocyanate  is  made  up  so  that  1  c.c.  exactly  corresponds  to  1 
c.c.  of  the  silver  nitrate.  Silver  thiocyanate  (white)  precipitates 
first.  When  all  the  silver  is  precipitated,  red  ferric  thiocyanate 
is  formed. 

Into  a  100  c.c.  volumetric  .flask  measure  accurately  with  a 
pipette  10  c.c.  urine  and  20  c.c.  standard  silver  nitrate  solution. 
Add  about  4  c.c.  cone,  nitric  acid  and  about  5  c.c.  iron  alum 
solution.  Add  distilled  water  up  to  the  100  c.c.  mark,  stopper 
and  mix  thoroughly.  Filter  through  a  dry  filter  into  a  dry 
vessel.  The  above  process  will  have  precipitated  all  chlorides 
as  AgCl,  and  an  excess  of  silver  nitrate  will  be  left  over. 

Accurately  measure  50  c.c.  of  the  filtrate  into  an  Erlenmeyer 
or  beaker  and  titrate  the  excess  of  silver  with  standard  thio- 
cyanate to  a  permanent  faint  pink  or  reddish-brown.  The 
amount  of  thiocyanate  used  corresponds  to  the  excess  of  silver 
remaining  in  the  solution. 

One  c.c.  thiocyanate  corresponds  to  1  c.c.  of  the  standard  sil- 
ver nitrate. 

One  c.c.  standard  silver  nitrate  corresponds  to  0.01  g  NaCl. 
Since  only  half  the  liquid  was  titrated,  multiply  the  titration 


298  PHYSIOLOGICAL   CHEMISTRY 

figure  by  two.     This  gives  the  excess  of  silver  nitrate  remain- 
ing. 

Subtract  the  excess  of  silver  solution  from  the  total  amount 
added.  This  will  give  the  amount  of  silver  solution  required 
to  precipitate  the  chlorides  present.  Since  each  cubic  centimeter 
of  silver  nitrate  will  precipitate  the  chloride  from  0.01  grams 
of  sodium  chloride,  the  sodium  chloride  in  10  c.e.  of  urine 
may  be  calculated.  From  this,  calculate  the  sodium  chloride  in 
the  24  hour  sample. 

14.  Sulphates. — 

The  most  accurate  and  satisfactory  methods  for  estimating 
sulphates  are  gravimetric.  (See  Folin's  methods.)  The  sul- 
phates are  precipitated  by  adding  barium  chloride,  the  precipi- 
tate collected  and  weighed.  Inorganic  sulphates  are  estimated 
directly.  Inorganic  +  ethereal,  after  the  splitting  of  ethereal 
sulphates  by  boiling  with  hydrochloric  acid.  Total  sulphur  is 
estimated  by  fusing  the  urine  with  an  oxidizing  agent  and 
determining  the  sulphates  as  above. 

Neubauer  has  suggested  the  following  approximate  volu- 
metric method  which  serves  for  most  clinical  purposes: 
Measure  50  c.c.  of  urine  into  a  flask,  add  3  c.c.  pure  HCl  and 
boil  gently  for  15  minutes  to  decompose  the  ethereal  sul- 
phates. From  a  burette  run  in  standard  barium  chloride  solu- 
tion (1  c.c.  =  0.01  gm.  SO3)  as  long  as  a  precipitate  forms, 
the  mixture  being  kept  hot.  After  running  in  the  first  3-4 
c.c.  of  barium  chloride,  allow  the  precipitate  to  settle  and 
with  a  glass  rod  remove  a  drop  of  the  liquid.  Place  it  on  a 
watch  glass  over  a  black  surface  and  add  a  few  drops  of  the 
BaClg  solution.  If  there  is  a  precipitate,  return  the  whole 
to  the  flask,  rinsing  in  the  last  traces  with  water,  and  add 
more  BaClg.  Again  allow  to  settle  and  test  as  before.  Pro- 
ceed until  no  more  BaS04  is  precipitated.  Excess  of  BaClg 
must  be  avoided.  When  the  minimal  excess  has  been  added, 
a  drop  of  the  clear  fluid  removed  from  the  flask  will  give  only 
a  cloudiness  with  a  drop  of  dilute  H2SO4.     If  more  than  a 


UEINE  299 

cloudiness  appears,  excess  has  been  added  and  the  whole 
operation  must  be  repeated.  Calculate  the  amount  of  SO3 
present  in  50  c.c.  urine  and  in  the  24  hour  sample. 

15.  Phosphates. — 

When  a  solution  of  disodium  phosphate  acidified  with  acetic 
acid  is  treated  with  a  solution  of  uranium  acetate,  a  white 
precipitate  of  uranium  phosphate  is  formed.  To  determine 
the  end  point,  a  drop  of  the  liquid  is  brought  in  contact  with 
a  drop  of  potassium  ferrocyanide  on  a  white  tile.  Any  excess 
of  uranium  causes  a  brown  color  to  appear. 

To  50  c.c.  of  urine  in  a  small  beaker,  add  5  c.c.  of  a  special 
sodium  acetate  mixture  (100  g.  sodium  acetate  dissolved  in 
800  c.c.  water  plus  100  c.c.  30%  acetic  acid  and  the  volume 
made  up  to  1  liter).  This  changes  any  acid  phosphate  into 
diaeid  sodium  phosphate.  Heat  to  boiling  and  from  a  burette 
run  in  drop  by  drop  a  standard  uranium  acetate  mixture 
(1  c.c.  is  equivalent  to  0.005  g.  of  P2O5).  Keep  the  mixture  at 
the  boiling  point.  From  time  to  time  remove  a  drop  with  a 
glass  rod  or  dropper  and  bring  it  in  contact  with  a  drop  of 
potassium  ferrocyanide  on  a  white  tile.  If  a  glass  rod  is 
used  it  should  be  rinsed  before  replacing  it  in  the  beaker.  It 
is  convenient  to  arrange  a  tile  with  several  drops  of  ferro- 
cyanide before  beginning  the  titration.  The  first  appearance 
of  brown  is  taken  as  the  end  point.  Calculate  the  weight  of 
P2O5  represented  by  the  phosphates  in  50  c.c.  of  urine,  and 
from  this  the  amount  in  the  24  hour  sample.  Calculate  what 
weight  of  phosphorus  this  represents. 

4.  Pathologic  Urine 

Some  substances  occur  in  pathological  urine  which  are 
absent  from,  or  found  only  in  traces  in  normal  urine.  Im- 
portant among  these  substances  are  various  proteins,  carbohy- 
drates, and  the  acetone  bodies. 

I.  Proteins. — The  proteins  most  frequently  found  are  albu- 
min,   globulin,   nucleo-protein,   hemoglobin,    glycoprotein;    of 


300  PHYSIOLOGICAL    CHEMISTRY 

the  protein  derivatives,  metaproteins,  proteoses,  peptones  and 
amino  acids. 

As  turbidity  would  interfere  with  the  detection  of  small 
amounts  of  protein,  the  urine  should  be  clear  before  testing. 
If  necessary,  filter,  repeating  the  filtration  and  adding  a 
small  amount  of  bismuth  subnitrate  if  the  filtrate  did  not 
come  through  clear.  If  the  urine  still  remains  cloudy,  add 
a  drop  or  two  of  barium  chloride  and  then  a  drop  or  two  of 
sodium  carbonate  solution.  Barium  carbonate  precipitates 
and  carries  down  with  it  the  suspended  material. 

a.  Albumins  and  Globulins, 

1.  Heat  Test. — The  heat  test  should  be  made  in  acid  solu- 
tion, as  only  in  a  weak  acid  solution  will  the  proteins  coagu- 
late properly  on  boiling.  The  urine  should  be  tested  with 
litmus  and  acidified  if  necessary  with  0.5%  acetic  acid.  Even 
if  the  urine  is  acid,  it  is  well  to  add  a  few  drops  of  dilute 
(0.5%)  acetic  acid  to  insure  coagulation  of  proteins  possibly 
present. 

Boil  a  few  cubic  centimeters  of  acidified  urine.  If  no  pre- 
cipitate forms,  albumin  and  globulin  are  absent.  If  it  becomes 
cloudy  or  a  precipitate  forms,  albumin  or  globulin  is  present. 

Do  not  add  acetic  acid  before  heating:  (1)  because  albumin, 
if  present,  may  be  converted  into  acid  albumin;  (2)  because 
acid  tends  to  disintegrate  cellular  elements  which  may  be 
present,  with  the  consequent  separation  of  cell-proteins.  The  use 
of  strong  mineral  acids  in  this  test  is  objectionable  for  simi- 
lar reasons. 

2.  Heller's  Nitric  Acid  Test. — To  four  c.c.  of  urine  in  a  test 
tube  add,  by  means  of  a  pipette,  a  few  cubic  centimeters  of 
concentrated  nitric  acid.  Run  in  carefully,  so  that  two  distinct 
layers  may  be  formed.  In  the  presence  of  albumin  or  globu- 
lin, a  distinct  white  ring  will  form  at  the  junction  of  the 
two  layers. 

The  following  important  facts  should  be  taken  into  con- 
sideration:   Not  over  five  minutes  should  be  occupied  in  this 


URINE  301 

test,  since  prolonged  action  of  nitric  acid  will  separate  cell- 
proteins,  which  may  be  present. 

When  urea  is  present  in  large  quantities,  particularly  if 
the  volume  of  the  urine  is  low,  crystals  of  urea  nitrate  may 
be  deposited  at  the  line  of  contact  of  the  two  liquids.  The 
crystalline  character  of  this  material  suffices  for  differentiation. 

In  the  presence  of  large  quantities  of  uric  acid,  a  whitish 
ring  may  appear  several  mm.  above  the  line  of  contact.  If 
doubt  exists  as  to  either  of  these  two  conditions,  dilute  the  urine 
and  repeat  the  test.  If  the  ring  Avas  due  to  uric  acid  or  urea,  it 
will  fail  to  appear  after  dilution.  A  cloud,  high  up  in  the  urine, 
may  be  caused  by  mucin.  The  pigments  form  rings  of  dark 
color  between  the  urine  and  the  acid.  These  rings,  once  recog- 
nized, cannot  be  mistaken  for  proteins.  A  ring  due  to  certain 
ingested  substances,  e.  g.,  copaiba,  may  be  dissolved  with  ether; 
iodine,  excreted  in  the  urine,  forms  a  dark  ring  at  the  surface 
of  the  nitric  acid.  Indican  is  the  most  common  pigment  produc- 
tive of  colored  rings. 

3.  Ferrocyanide  Test. — Add  a  few  drops  of  potassium  ferrocy- 
anide  solution  to  the  clear  urine  and  then  acidify  with  acetic  acid. 
In  the  presence  of  albumin  or  globulin,  a  white  cloud  or  ring 
will  be  formed.  Observed  in  a  strong  light,  this  will  be  seen  to 
be  of  a  floeculent  character. 

4.  Quantitative  Estimation,  (Approximate.) — Pour  the  urine 
into  an  Esbach  tube  up  to  the  mark  ' '  urine  "  or  U.  Add  Esbach  's 
reagent  (2%  citric  acid,  1%  picric  acid)  to  the  mark  "reagent" 
or  R,  close  the  tube  carefully  with  a  stopper  and  invert  several 
times  until  the  fluids  are  well  mixed.  Allow  the  tube  to  stand  24 
hours.  Read  on  the  scale  the  amount  of  the  precipitate.  The 
scale  corresponds  to  grams  protein  per  1000  c.c.  of  urine. 

Although  this  method  gives  only  approximate  results  it 
usually  is  sufficient  for  clinical  purposes. 

5.  Serum  Glohulin  Differentiation. 

(a)  Make  a  few  cubic  centimeters  of  clear  urine  alkaline  with 
ammonia.  Filter  off  the  precipitated  phosphates.  Neutralize 
the  filtrate  with  acetic  acid  and  treat  with  an  equal  volume 


302  PHYSIOLOGICAL   CHEMISTRY 

of  saturated  ammonium  sulphate  solution.  Globulin,  if  pres- 
ent, will  be  thrown  down.  If  the  urine  is  rich  in  urates  they 
may  be  precipitated  but  may  be  recognized  with  the  microscope 
or  by  the  murexid  test. 

(b)  To  a  few  cubic  centimeters  of  clear  urine,  add  an  excess 
of  solid  magnesium  sulphate.  Shake  thoroughly  and  globu- 
lins, if  present,  will  be  precipitated  and  will  be  deposited  above 
the  excess  of  salt. 

(c)  Add  a  few  drops  of  clear  urine  to  a  large  beaker  full 
of  distilled  water.  If  globulins  are  present,  a  white  cloud  will 
appear. 

b.  NUCLEOPEOTEIN. 

After  ascertaining  that  the  clear  urine  does  not  coagulate 
upon  heating,  dilute  a  small  amount  with  water  in  equal  quan- 
tities, to  prevent  the  precipitation  of  uric  acid  and  to  reduce 
the  solvent  action  of  the  salts  upon  the  nucleoprotein.  Add 
acetic  acid  drop  by  drop,  Nucleoprotein,  if  present,  will  be  pre- 
cipitated. 

Nucleoproteins  often  are  present  in  large  quantities  in 
various  forms  of  proteinuria,  such  as  physiologic  proteinuria 
(occurring  at  times  in  healthy  individuals  after  excessive  exer- 
tion, etc.),  orthostatic  proteinuria  (a  form  in  which  the  pro- 
tein disappears  from  the  urine  if  the  patient  is  kept  lying 
down),  and  lordotic  proteinuria  (a  form  accompanying  lordo- 
sis, a  variety  of  spinal  curvature). 

It  should  be  borne  in  mind,  however,  that  the  nucleoprotein 
may  have  its  origin  from  disintegrated  cells,  which  may  be 
present  in  large  amount. 

c.  Metapeotein. 

The  proteins  present  in  the  urine,  may  be  converted,  by  acids 
or  alkali  present,  into  the  metaproteins.  Ascertain  the  chem- 
ical reaction  of  urine  and  neutralize  clear  urine  carefully.  If 
precipitation  occurs,  heat;  the  derived  albumin  will  be  coagu- 
lated and  will  not  redissolve  on  adding  acid  or  alkali. 


URINE  303 

a.  Proteoses  and  Peptones. 

These  forms  may  appear  in  the  urine  occasionally. 

1.  Neutralize  and  boil  the  clear  urine  to  remove  coagulable 
proteins.  Filter  and  to  the  filtrate,  slightly  warm,  add  a  few 
drops  of  potassium  ferrocyanide  solution  and  10%  acetic  acid. 
If  a  precipitate  appears,  it  is  proteose.  Heat  and  it  will  dis- 
appear; cool  and  it  will  reappear. 

2.  To  clear  urine,  previously  acidified  and  boiled,  add  ex- 
cess of  solid  ammonium  sulphate  and  a  few  drops  of  acetic 
acid;  proteoses,  if  present,  will  be  precipitated.  Filter.  Dis- 
solve the  precipitate  in  distilled  water  and  apply  the  biuret 
test.  Ammonium  sulphate  interferes  with  the  reaction  and 
must  be  removed  with  barium  carbonate, 

3.  A  specimen  of  urine  may  be  dialyzed  and  the  diffusate 
tested  for  peptones  with  the  biuret  test. 

II.  Carbohydrates. — 

The  carbohydrates  most  frequently  found  in  the  urine  are 
dextrose  and  lactose.  In  addition  to  these,  pentoses,  levulose, 
galactose,  and  saccharose  may  occur.  These  substances  are 
detected  by  the  usual  reduction,  phenylhydrazine  and  fermen- 
tation tests,  or  by  the  optical  activity  of  the  urine  containing 
them.  For  differentiating  among  the  various  carbohydrates, 
the  methods  employed  are  those  already  familiar  to  the  student 
from  his  study  of  the  carbohydrates. 

With  samples  of  the  carbohydrate  urine  furnished  perform 
the  following  tests: 

a.  Reduction  Tests. — Albumin  or  globulin  must  be  removed 
if  present  by  acidifying  slightly  with  acetic  acid,  boiling  and 
filtering. 

1.  Qualitative  Test. — Perform  the  Fehliug  test.  The  test  is 
subject  to  two  sources  of  error :  unless  the  liquid  is  boiled  the  test 
is  not  sensitive;  if  boiled,  other  substances  such  as  uric  acid, 
creatinine,  mucin,  pentoses,  glycuronic  acids,  lactose,  or  a  reduc- 
ing agent  used  in  the  preservation  of  the  urine,  such  as  chloro- 
form or  formaldehyde  may  give  a  reduction. 


304  PHYSIOLOGICAL    CHEMISTRY 

2.  Quantitative  Estimation  of  Sugar. — The  quantitative  esti- 
mation of  sugar  in  urine  is  attended  by  some  difficulty,  and  a 
variety  of  methods  have  been  proposed.  Only  two  titration 
methods  will  be  included  here,  first  the  Fehling  method,  because 
it  is  the  standard  method  upon  which  most  later  and  more  ac- 
curate methods  are  based,  and  second  Benedict's  method,  which 
is  perhaps  the  best  for  general  use,  all  things  considered. 

(a)  Fehling  Method. — The  Fehling  test  may  be  made  quan- 
titative by  making  up  the  copper  sulphate  solution  accurately. 
A  given  amount  of  dextrose  will  reduce  a  definite  amount  of 
copper.  As  long  as  copper  sulphate  remains  unreduced  the 
solution  will  have  a  blue  color.  A  measured  volume  of  Feh- 
ling's  solution  may  thus  be  titrated  with  the  dextrose  urine 
until  the  color  has  completely  disappeared.  From  the  volume 
of  urine  necessary  to  reduce  the  given  volume  of  Fehling 's 
solution,  the  amount  of  dextrose  in  the  urine  may  be  calculated. 

The  quantitative  Fehling  solution  is  made  up  of  such  a 
strength  that  10  c.c.  of  the  copper  solution  (20  c.c.  of  the 
mixed  Fehling 's)  will  be  reduced  by  0.1  gm.  glucose  or  0.134 
gm.  lactose. 

Before  starting  the  experiment  read  through  the  entire  direc- 
tions for  the  process. 

"With  a  pipette  measure  10  c.c.  of  each  part  of  the  quantita- 
tive Fehling 's  solution  into  an  Erlenmeyer  flask  (or  evaporat- 
ing dish).  Add  80  c.c.  of  water  measured  with  a  graduate. 
Dilute  5  c.c.  of  urine  (measured  with  a  pipette)  with  20  c.c. 
distilled  water  (also  measured  with  a  pipette).  Diluting  in  a 
volumetric  flask  is  preferable,  but  the  method  described  is 
satisfactory. 

Fill  a  burette  with  the  diluted  urine.  Heat  the  diluted 
Fehling 's  solution  in  the  Erlenmeyer  with  a  small  flame,  and 
run  in  the  urine,  first  a  few  drops  at  a  time,  boiling  a  few 
seconds  after  each  addition.  If  the  liquid  remains  blue  after 
the  addition  of  2  c.c.  of  urine,  continue  the  process,  adding  the 
urine  in  0.2  -0.3  c.c.  portions  until  the  blue  or  green  color  has 
almost  disappeared,  then  drop  by  drop  until  no  blue  or  green 


URINE  305 

tinge  can  be  detected.  Observe  over  a  white  paper.  Do  not 
look  through  the  liquid  at  the  blue  sky.  The  volume  of  urine 
required  to  reduce  the  20  c.c.  of  Fehling's  will  contain  0.1  gm, 
dextrose  or  0.134  gm.  lactose.  Perform  duplicate  analyses. 
Time  may  be  saved  by  running  through  a  trial  titration  before 
the  actual  determination,  in  order  to  find  roughly  the  volume 
of  urine  required  to  reduce  the  Fehling's  solution. 

Calculate  the  weight  of  sugar  in  100  c.c.  of  urine.  If  the 
sugar  content  of  the  urine  is  so  high  that  less  than  2  c.c,  are 
required  to  reduce  20  c.c.  of  Fehling's  solution,  the  urine  must 
be  further  diluted. 

(b)  Benedict's  Method. — The  greater  accuracy  of  this  method 
is  due  to  several  facts:  the  solution  is  less  strongly  alkaline,  so 
that  the  decomposition  of  the  sugar  on  boiling  is  less,  and  the 
end  point  is  perhaps  sharper  than  in  the  original  Fehling 
method. 

Standard  Copper  Solution. — Eighteen  grams  pure  crystalline 
CuSO^ ;  200  grams  crystalline  NaaCOg ;  200  grams  sodium  or 
potassium  citrate;  125  grams  KCNS;  5  c.c.  of  a  5%  solution  of 
K4Fe(CN)6;  distilled  water  to  make  a  total  volume  of  1  liter. 
Dissolve  the  carbonate,  citrate  and  thiocyanate  in  about  700 
c.c.  of  water  and  filter  if  necessary.  Dissolve  the  copper  sul- 
phate in  100  c.c.  of  water,  and  add  slowly,  stirring  constantly, 
to  the  700  c.c.  Add  the  ferrocyanide  and  make  up  to  exactly 
1  liter.  The  only  ingredient  which  need  be  weighed  exactly 
is  the  copper  sulphate. 

Analysis. — Measure  25  c.c.  (pipette)  of  the  reagent  into  a  25-30 
cm.  evaporating  dish.  Add  10-20  grams  crystalline  NagCOa,  or 
%  this  amount  of  the  anhydrous  salt,  some  talcum  or  powdered 
pumice  and  heat  to  boiling  over  a  free  flame  until  the  carbonate 
has  dissolved. 

Accurately  dilute  10  c.c.  of  urine  to  100  c.c.  unless  the 
amount  of  sugar  in  it  is  small,  when  it  can  be  used  without 
dilution.  Fill  a  burette  with  the  urine,  and  run  it  into  the 
boiling  copper  solution,  rapidly  at  first  and  then  more  slowly 
as  the  color  grows  less,  then  a  few  drops  at  a  time  until  the 


306  PHYSIOLOGICAL    CHEMISTRY 

solution  is  colorless.  A  white  precipitate  forms  during  the 
titration.  If  the  liquid  in  the  dish  becomes  too  concentrated, 
add  water. 

Calculation. — Exactly  50  mg.  of  glucose  will  reduce  the  25  c.c. 
of  the  copper  reagent.  This  amount  of  glucose  must  have  been 
present  in  the  volume  of  urine  used,  provided  no  other  reducing 
substances  were  present.  If  the  urine  was  diluted  10  times, 
then  the  per  cent  of  glucose  in  the  original  urine  was 
0.050  X  1000  where  s  is  the  volume  of  urine  used  from  the 

X 

burette. 

b.  Fermentation  Tests. 

Perform  a  fermentation  test  on  carbohydrate  urine. 

This  test  is  a  very  satisfactory  one,  as  the  other  reducing  sub- 
stances in  the  urine  do  not  ferment.  It  furnishes  also  a  means 
of  differentiation  between  dextrose  and  lactose  as  certain 
varieties  of  yeast  will  ferment  dextrose  but  not  lactose. 

c.  Phenylhydrazine  Test. 

This  test  is  carried  out  as  described  under  carbohydrates,  and 
may  be  used  to  distinguish  various  members  of  the  group. 

d.  Optical  Activity. 

In  testing  for  sugars  with  the  polariscope  it  must  be  remem- 
bered that  other  substances,  such  as  proteins,  etc.,  may  be  the 
cause  of  any  observed  rotation. 

III.  Acetone  Bodies. — 

The  term  ''acetone  bodies"  includes  acetone,  ;8-oxybutyric 
acid  and  acetoacetic,  and  this  is  readily  converted  into  acetone 
by  the  loss  of  CO2,  so  that  these  acids  are  never  found  in  the 
urine  unaccompanied  by  acetone.  Acetone  may  occur,  how- 
ever, when  these  acids  are  not  present. 

a.  Acetone,  if  present  in  large  amounts,  may  be  tested  for 
in  the  urine  directly.  If  only  small  amounts  are  present,  the 
urine  may  be  distilled.     The  acetone  will  go  over  in  the  first 


URINE  307 

few  cubic  centimeters  of  distillate.     An  acetone  urine  wiU  be 
supplied  containing  sufficient  acetone  to  give  the  tests. 

1.  Rotliera's  Test. — Saturate  about  10  c.c.  of  urine  with  am- 
monium sulphate.  Add  2-3  drops  of  fresh  sodium  nitroprusside 
solution  and  2-3  c.c.  concentrated  ammonia.  Mix  and  allow 
to  stand  at  least  ^  hour,  A  characteristic  permanganate 
color  indicates  the  presence  of  acetone. 

2.  Iodoform  Test. — Perform  the  iodoform  test  with  acetone 
urine.    (See  under  Fermentation.) 


APPENDIX 


DIRECTIONS  FOR  MAKING  UP   QUANTITATIVE 
OR  SPECIAL  REAGENTS 

Ammonium  Thiocyanate,  Standard,  for  Chlorides. — 

One  c.c.  is  equivalent  to  1  c.c.  standard  AgNOg. 

This  solution  is  made  of  such  a  strength  that  1  c.c.  of  it 
is  equal  to  1  c.c.  of  the  standard  silver  nitrate  solution  men- 
tioned below.  To  prepare  the  solution  dissolve  12.9  grams 
of  ammonium  thiocyanate,  NH^SCN,  in  a  little  less  than  a 
liter  of  water.  In  a  small  flask  place  20  c.c.  of  the  standard 
silver  nitrate  solution,  5  c.c.  of  a  cold  saturated  solution  of 
ferric  alum  and  4  c.c.  of  nitric  acid  (sp.  gr.  1.2),  add  water 
to  make  the  total  volume  100  c.c,  and  thoroughly  mix  the 
contents  of  the  flask.  Now  run  in  the  ammonium  thiocyanate 
solution  from  a  burette  until  a  permanent  red-brown  tinge 
is  produced.  This  is  the  end-reaction  and  indicates  that  the 
last  trace  of  silver  nitrate  has  been  precipitated.  Take  the 
burette  reading  and  calculate  the  amount  of  water  neces- 
sary to  use  in  diluting  the  ammonium  thiocyanate  in  order 
that  10  c.c.  of  this  solution  may  be  exactly  equal  to  10  c.c. 
of  the  silver  nitrate  solution.  Make  the  dilution  and  titrate 
again  to  be  certain  that  the  solution  is  of  the  proper  strength. 

Barfoed's  Solution. — 

Dissolve  4.5  grams  of  neutral,  crystallized  copper  acetate 
in  100  c.c.  of  water  and  add  1.2  c.c.  of  50%  acetic  acid. 

Barium  Chloride  for  Sulphate  Determination. — 

Thirty  and  five-tenths  grams  BaClg .  2H2O  made  up  to  1  liter 
with  distilled  water. 

One  c.c.  corresponds  to  0.01  grams  SO3. 

308 


APPENDIX  309 

Benedict's   Solution  for   Carbohydrate  Estimation. — 

See  under  description  of  the  method  in  the  laboratory  direc- 
tions. 

Congo  Paper. — 

Dissolve  1  gram  of  Congo  red  in  90  c.c.  water,  add  10 
c.c.  95%  alcohol.  Dip  filter  paper  into  this  solution,  and 
allow  to  dry. 

Esbach's  Reagent. — 

Dissolve  10  grams  of  picric  acid  and  20  grams  of  citric 
acid  in  1  liter  of  water. 

Fehling's  Solution.    (Quantitative.) — 

Fehling's  solution  is  composed  of  two  definite  solutions — 
a  copper  sulphate  solution  and  an  alkaline  tartrate  solution, 
which  may  be  prepared  as  follows: 

A.  Copper  sulphate  solution  =  34.65  grams  of  copper  sul- 
phate dissolved  in  water  and  made  up  to  500  c.c. 

B.  Alkaline  tartrate  solution  =  125  grams  of  potassium  hy- 
droxide and  173  grams  of  Eochelle  salt  dissolved  in  water 
and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber- 
stoppered  bottles  and  mixed  in  equal  volumes  when  needed 
for  use.     This  is  done  to  prevent  deterioration. 

Only  the  copper  salt  need  be  weighed  on  the  quantita- 
tive balance. 

Fehling's  Solution.     (Qualitative.) — 

Amounts  as  in  quantitative,  but  weighed  on  the  ordinary 
balance. 

Folin-Schaffer  Reagent. — 

This  reagent  consists  of  500  grams  of  ammonium  sulphate, 
5  grams  of  uranium  acetate,  and  60  c.c.  of  10%  acetic  acid  in 
650  c.c.  of  distilled  water. 

Formalin.     (Neutral.) — 

Dilute    formalin    1-4    with    distilled    water,    add    a    small 


310  PHYSIOLOGICAL   CHEMISTRY 

amount  of  phenolphthalein,  and  then  add  dilute  (N/10)  sodium 
hydrate  until  the  liquid  acquires  a  faint  pink  tinge. 

Glyoxylic  Acid  Solution. — 

Hopkins-Cole  Eeagent  (Benedict's  Modification). — Ten 
grams  of  powdered  magnesium  are  placed  in  a  large  Erlen- 
meyer  flask  and  shaken  up  with  enough  distilled  water  to 
liberally  cover  the  magnesium.  Two  hundred  and  fifty  cubic 
centimeters  of  a  cold,  saturated  solution  of  oxalic  acid  is 
now  added  slowly.  The  reaction  proceeds  very  rapidly  and 
with  the  liberation  of  much  heat,  so  that  the  flask  should  be 
cooled  under  running  water  during  the  addition  of  the  acid. 
The  contents  of  the  flask  are  shaken  after  the  addition  of 
the  last  portion  of  the  acid  and  then  poured  upon  a  filter, 
to  remove  the  insoluble  magnesium  oxalate.  A  little  wash 
water  is  poured  through  the  filter,  the  filtrate  acidified  with 
acetic  acid  to  prevent  the  partial  precipitation  of  the  mag- 
nesium on  long  standing,  and  made  up  to  a  liter  with  dis- 
tilled water.  This  solution  contains  only  the  magnesium  salt 
of  glyoxylic  acid. 

Guenzberg's  Reagent. — 

Dissolve  2  grams  of  phlorglucinol  and  1  gram  of  vanillin 
in  100  c.c.  of  95%  alcohol. 

Magnesia  Mixture. — 

Dissolve  175  grams  of  magnesium  sulphate  and  350  grams 
of  ammonium  chloride  in  1,400  c.c.  of  distilled  water.  Add 
700  grams  of  concentrated  ammonium  hydroxide,  mix  thor- 
oughly, and  preserve  the  mixture  in  a  glass-stoppered  bottle. 

Mett's  Tubes.— 

See  directions  for  making  in  chapter  on  gastric  digestion. 

Millon's  Reagent. — 

Digest  1  part  (by  weight)  of  mercury  with  2  parts  (by 
weight)  of  nitric  acid  (sp.  gr.  1.42)  and  dilute  the  resulting 
solution  with  2  volumes  of  water. 


APPENDIX  311 

Molisch's  Reagent.— 

A  15%  alcoholic  solution  of  oc  naphthol. 

Nylander's  Reagent. — 

Digest  2  grams  of  bismuth  subnitrate  and  4  grams  of 
Rochelle  salt  in  100  c.c.  of  a  10%  solution  of  potassium  hy- 
droxide.   The  reagent  should  then  be  cooled  and  filtered. 

Pancreatic  Solution.    ("Artificial  pancreatic  juice.") — 

One  per  cent  neutral  solution  of  pancreatic  powder  in 
water. 

Pepsin  Solution.    (Artificial  gastric  juice.) — 

One  per  cent  solution  of  pepsin  flakes  in  0.2%  HCl. 

Potassium  Bichromate  N/2. — 

Dissolve  24.55  grams  pure  potassium  bichromate  in  distilled 
water  and  make  up  to  1  liter  of  solution. 

Potassium  Permanganate  N/20. — 

Dissolve  1.578  grams  of  the  pure  salt  in  distilled  water 
and  make  up  to  1  liter. 

Potassium  Pyroantimonate  K^'H.^Slo^O^. — 

Two  grams  of  KgHaSbgOy  are  added  to  100  c.c.  of  boiling 
water,  the  mixture  is  boiled  until  the  salt  is  dissolved,  and  the 
solution  quickly  cooled.  Three  c.c.  of  10%  KOH  solution  is 
added  to  it  to  render  the  reagent  alkaline.     (Bray.) 

Silver  Nitrate  (Standard)  for  Volhard  Chloride  Method.— 

One  c.c.  is  equivalent  to  0.01  grams  NaCl. 
Dissolve  29.042  grams  of  pure  silver    nitrate    in    distilled 
water  and  make  up  to  1  liter. 

Sodium  Cobaltinitrite  NagCo  (N02)6- — 

One  hundred  grams  of  NaNOg  are  dissolved  in  200  c.c.  of 
water,  and  to  this  solution  50  c.c.  of  6-molar  acetic  acid  and 
10  grams  of  CoCNOg),  .6  aq.  are  added.    After  a  day  or  two  the 


312  PHYSIOLOGICAL    CHEMISTRY 

solution  is  filtered  from  any  precipitate,  KsNa  [Co(N02)e].  aq. 
and  diluted  to  400  CO.     (Bray.) 

Special  Sodium  Acetate  Solution  (for  Uranium  Acetate 
Method  for  Phosphates.) — 

Dissolve  100  grams  of  sodium  acetate  in  800  c.c.  of  distilled 
water,  add  100  c.c.  of  30%  acetic  acid  to  the  solution,  and 
make  the  volume  of  the  mixture  up  to  1  liter  with  distilled 
water.  ^ 

Stokes'  Reag-ent. — 

A  solution  containing  2%  ferrous  sulphate  and  3%  tar- 
taric acid.  "When  needed  for  use  a  small  amount  should  be 
placed  in  a  test  tube  and  ammonium  hydroxide  added  until 
the  precipitate  which  forms  on  the  first  addtiion  of  the  hy- 
droxide has  entirely  dissolved.  This  produces  ammonium 
ferrotartrate,  which  is  a  reducing  agent. 

Toepfer's  Reagent. — 

Dissolve  0.5  grams  of  dimethylamino-azobenzene  in  100  c.c. 
of  95%  alcohol. 

Uranium  Acetate  Solution  (Standard). — 

Dissolve  about  35.0  grams  of  uranium  acetate  in  1  liter 
of  water  with  the  aid  of  heat  and  3-4  c.c.  of  glacial  acetic 
acid.  Let  stand  a  few  days  and  filter.  Standardize  against 
a  phosphate  solution  containing  0.005  gram  of  PgOg  per  cubic 
centimeter.  For  this  purpose  dissolve  14.721  grams  of  pure 
air-dry  sodium  ammonium  phosphate  (NaNH4HP04 .  4H2O) 
in  water  to  make  a  liter.  To  20  c.c.  of  this  phosphate  solution 
in  a  200  c.c.  beaker  add  30  c.c.  of  water  and  5  c.c.  of  sodium 
acetate  solution  (see  above)  and  titrate  with  the  uranium 
solution  to  the  correct  end  reaction  as  indicated  in  the  method 
proper.  If  exactly  20  c.c.  of  uranium  solution  are  required, 
]  c.c.  of  the  solution  is  equivalent  to  0.005  gram  P2O5.  If 
stronger  than  this,  dilute  accordingly  and  check  again  by 
titration. 


APPENDIX 


313 


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REFERENCES 


The  following  list  will  furnish  the  student  with  a  nucleus  of  reference 
material.  Current  journals  such  as  the  Journal  of  Biological  Chemistry,  the 
Biochemical  Journal,  and  Chemical  Abstracts  sh-ould  be  consulted.  The 
ten-year  index  of  the  last  named  journal  is  now  being  issued,  and  when 
completed  will  serve  as  an  invaluable  survey  of  biochemical  work  from 
1907  to  1916. 

General  Texts  or  Reference  Works 

Hammarsten-Mandel  :     Textbook  of  Physiological  Chemistry,  John  Wiley 

&  Sons,  New  York,  1914. 
Matthews  :   Physiological  Chemistry,  William  Wood  &  Co.,  New  York,  1916. 
Abderhalden:     Lehrbuch  der  Physiologischen  Chemie,  1914,  1915. 
Von  Furth:     Probleme  der  Phys.  u.  Path.  Chemie,  Vogel,  Leipzig,  1912. 
Abderhalden:      Biochemisches    Handlexicon,   Springer,    Berlin.     A   large 

work  of  reference  in  several  volumes. 
Czapek:     Biochemie  der  Pflanzen,  G.  Fischer,  Berlin,  1913. 
Barger:     The  Simpler  Natural  Bases    (Plimmer  Monograph),  Longmans, 

Green  &  Co.,  New  York,  1914. 

For  Lalioratory  Methods 

Abderhalden:     Handbueh  der  Bioehemischen  Arbeitsmethoden,  Urban  and 
Schwarzenburg.     A  large  work  in  many  volumes. 

Hawk:     Practical  Physiological  Chemistry,  P.  Blakiston's  Son  &  Co.,  Phila- 
delphia, 1916. 

Plimmer:     Practical  Organic  and  Biochemistry,  Longmans,  Green  &  Co., 
New  York,  1915. 

Findlay:     Osmotic  Pressure,  Longmans,  Green  &  Co.,  New  York,  1913. 

Pats 

Leathes  :     The  Fats,  Longmans,  Green  &  Co.,  New  York,  1910. 

Carljoliydrates 

Armstrong:     Simple  Sugars  and  Glucosides,  Longmans,  Green  &  Co.,  New 
York. 

Nep,   J.   U. :       Dissoziationsvorgange   in   der   Zuckergruppe,   Annalen    der 
Chemie,  1913,  403,  204. 

Enzymes 

Harden:    Alcoholic  Fermentation  (Plimmer  Monograph),  Longmans,  Green 
&  Co.,  New  York,  1911. 

Euler:     The  General  Chemistry  of  Enzymes,  Trans,  by  Pope,  1912. 

COHNHEIM :     Enzymes,  John  Wiley  &  Sons,  New  York,  1912. 

315 


316  REFEEENCES 

Oppenheimer:     Die  Fermente  u.  ihre  Wirkungen,  Vogel,  Leipzig,  1913. 
Bayliss:     Nature  of  Enzyme  Action    (Plimmer  Monograph),  Longmans, 
Green  &  Co.,  New  York,  1914. 

Proteins 

Schryver:      General  Character  of  Proteins    (Plimmer  Monograph),  Long- 
mans, Green  &  Co.,  New  York,  1909. 

Mann:     Chemistry  of  the  Proteids,  Macmillan  Co.,  New  York. 

Osborne:      The    Vegetable    Proteins    (Plimmer    Monograph),    Longmans, 
Green  &  Co.,  New  York,  1912. 

Plimmer:     Chemical  Constitution  of  the  Proteins,  Longmans,  Green  &  Co., 
New  York,  Part  I,  1912;  Part  II,  1913. 

XJnderiiill:     Physiology  of  the  Amino  Acids,  Yale  University  Press. 

KossEL :     Protamines  and  Histones,  Longmans,  Green  &  Co.,  New  York,  • 
1914. 

Jones:      Nucleic  Aeida   (Plimmer  Monograph),  Longmans,   Green   &  Co., 
New  York,  1914. 

See  also  a  list  of  54  references  in  Matthews,  pages  187-189  (1916  edition). 

Colloids 

Ostwald:     Grundriss  der  Colloid  Chemie,  1909. 

Taylor:     Chemistry  of  Colloids,  Longmans,  Green  &  Co.,  New  York,  1915. 

Hatschek:      Introduction   to   the   Physics   and   Chemistry   of   Colloids,    P. 

Blakiston's  Son  &  Co.,  Philadelphia,  1916. 

roods 
Composition  of  Foods,  U.  S.  Dept.  of  Agriculture,  O.  E.  S.  Bull.  No.  221, 

1909. 
Leach:     Food  Inspection  and  Analysis,  John  Wiley  &  Sons,  New  York, 

1906. 
Mendel:     Changes  in  the  Food  Supply,  Yale  University  Press,  1916. 

Brain 

Levene  and  Jacobs:     Cerebrosides  of  Brain,  Journal  Biological  Chemistry, 
1912,  xii,  381  and  389. 

Blood 

Van  Slyke  and  Meyer:      Amino   Acid  Nitrogen  of   the  Blood,  Journal 
Biological  Chemistry,  1912,  xii,  399. 

Abdekhalden:      Amino    Acids    in    Blood,    Zeitschrift    fiir    physiologische 
Chemie,  1913,  Ixxxviii,  480. 

Abderhalden:      Abwehrfermente,   1914. 

Von  Hess  and  McGuigan:     Sugar  Content  Determined  by  Vivi-diffusion, 
Journal  Pharmacology  and  Experimental   Therapeutics,   1914,  vi. 

Abel:      Vivi-diffusion,    Journal    Pharmacology    and    Experimental    Thera- 
peutics, 1914,  iv,  611. 


REFERENCES  317 

Gradwohl-Blaivas  :  The  Newer  Methods  of  Blood  and  Urine  Chemistry, 
C.  V.  MoslDy  Co.,  St.  Louis,  1917. 

Digestion 

Beaumont:     Physiology  of  Digestion,  1847. 

Pavlov:     The  Work  of  the  Digestive  Glands,  Trans,  by  Thompson,  1910. 

Babkin:  Die  Aiissere  Sekretion  der  Verdauungsdriisen,  Springer,  Berlin, 
1914. 

Bernard:     Memoire  sur  le  pancreas,  Paris,  1856. 

Bayliss  and  Starling:    Enterokinase,  Journal  of  Physiology,  1903,  xxx,  61. 

Bayliss  and  Starling:     Secretin,  Journal  of  Physiology,  1902,  29  and  31. 

Carlson,  et  al.  :     Journal  American  Medical  Association,  1916,  Ixvi,  178. 

Wohlgemuth:  Composition  of  Pancreatic  Juice,  Biochemische  Zeitschrift, 
1912,  xxxix,  321. 

Mellanby  and  Woolley:  Trypsin  and  Trypsinogen,  Journal  of  Physi- 
ology, 1914,  xlvii,  339. 

Mellanby  and  Woolley:  Ferments  of  the  Pancreas,  Journal  of  Physiol- 
ogy, 1915,  xlix,  246. 

Hammarsten:     Bile,  Ergebnisse  der  Physiologic,  1905. 

Pregl:     Cholic  Acid,  Zeitschrift  fiir  physiologische  Chemie,  1910,  Ixv,  157. 

Lif.chutz:  Cholic  Acid,  Berichte  der  deutschen  chemischen  Gesellschaft, 
1914,  xlvii,  1459. 

Fischer  and  Rose:  Composition  of  Bilirubin,  Zeitschrift  fiir  physiol- 
ogische Chemie,  1914,  Ixxxix,  262, 

Grindley  and  MacNeal:  Bacteria  and  Chemistry  of  Feces  of  Healthy 
Men,  Studies  in  Nutrition,  Vols.  3,  4,  and  5,  University  of  Illinois, 
1912. 

Urine 

Neuberg:      Der  Harn,  Springer,  Berlin,  1911. 

FOLIN:  Laws  Governing  the  Chemical  Composition  of  the  Urine,  Amer- 
ican Journal  Physiology,  1905,  xiii,  66,  45;  Also  numerous  other  pa- 
pers by  Folin  in  the  Zeitschrift  fiir  Physiologische  Chemie,  Journal 
of  Biological  Chemistry,  American  Journal  of  Physiology,  and  else- 
where. 

Gradwohl-Blaivas  :  The  Newer  Methods  of  Blood  and  Urine  Chemistry, 
C.  V.  Mosby  Co.,  St.  Louis,  1917.  A  short  volume  giving  some  of  the 
more  recent  analytical  methods. 

Metabolism 

The  literature  of  metabolism  is  enormous.  The  following  brief  list 
will  serve  as  the  handle  of  the  fan,  from  which  the  usually  abundant  ref- 
erences will  lead  out  into  the  broader  or  more  specialized  aspects  of  the 
field. 


318  REFERENCES 

Energy  Exchange 

Lavoisier:     Publications  on  Animal  Heat,  Histoire  et  Memoires  de  PAcad. 
de  Sci.,  Paris,  1777-1789. 

Rubnee:     Zeitschrift  fur  Biologie,  1894,  xxx,  73. 

Atwater  and  Benedict:  Metabolism  of  Matter  and  Energy  in  the  Human 
Body,  U.  S.  Dept.  Agriculture  O.  E.  S.  Bull.,  No.  136,  1903. 

EussELL  Sage  Institute  of  Pathology:     Clinical  Calorimetry,  1915-17. 
(Papers  by  Lusk,  du  Bois,  Benedict  and  others.) 

G-eneral 

Von  Noorden:    Metabolism  and  Practical  Medicine,  Keener,  Chicago,  1907. 

Lusk:     The  Science  of  Nutrition,  W.  B.  Saunders  Co.,  1912. 

Von  Furth:    Probleme  der  Physiologischen  u.  Pathologischen  Chemie,  1912. 

Cathcart:     The  Physiology  of  Protein  Metabolism  (Plimmer  Monograph), 
Longmans,  Green  &  Co.,  New  York,  1912. 

Taylor:     Digestion  and  Metabolism,  Lea  &  Febiger,  Philadelphia,  1912. 

Dakin:     Oxidations  and  Eeductions  in  the  Animal  Body  (Plimmer  Mono- 
graph), Longmans,  Green  &  Co.,  New  York,  1912. 

Allen:     Glycosuria  and  Diabetes,  1913. 

Paton:     Nervous  and  Chemical  Regulator  of  Metabolism,  Macmillan  Co., 
New  York,  1913. 

McCay:     The  Protein  Element  in  Nutrition,  Longmans,  Green  &  Co.,  New 
York,  1912. 

Fletcher:     The  A  B  C  of  Our  Own  Nutrition. 

Albu-Neuberg  :      Mineral  Stoffwechsel,  Springer,  Berlin,  1906. 

Vincent:     Liternal  Secretion  and  the  Ductless  Glands,  Arnold,  London,1913. 

Benedict:     The  Influence  of  Inanition  on  Metabolism,  Carnegie  Institute 
Report,  Washington,  1907. 

Benedict:     Experiment  on  a  Man  Fasting  Thirty-one  Days,  Carnegie  In- 
stitute, Pub.  203,  1915. 

Lusk:     Fundamental  Basis  of  Nutrition,  Yale  University  Press,  1915. 

Lectures  on  Nutrition,  Journal  of  the  Washington  Academy  of   Science, 
1916,  vi. 

Vltamines 

Funk:     Lancet,  London;  Journal  of  Physiology;   and  Biochemical  Jour- 
nal from  1911  on. 

Numerous  papers  by  Osborne,  Mendel,  McCollum  and  others,  mostly  in 
the  Journal  of  Biological  Chemistry,  1912  on. 

Funk:     Review,  References  to  literature,  American  Medicine,  1916,  xi,  751. 

Voegthin:      Review,  Journal  Washington  Academy  of  Science,  1916,  vi, 
575;   Scientific  Alonthly,  1916,  ii,  289. 

Chamberlain:     Prevention  of  Beriberi  among  Philippine  Scouts,  Journal 
American  Medical  Association,  1915,  Ixiv,  1215. 


INDEX 


(See  also  separate  index  for  laboratory  work) 


Abderhalden   reaction,   123 
Absorption,  152-154 
general,  152 
in  intestine,   152 
in  mouth,   152 
of  carbohydrates,   153 
of  fats,   154 
of  proteins,  153 
spectra,  50 
Acetone  bodies,  in  diabetes,  190 
Acidity  of  gastric  contents,  134 

in  disease,  136 
Acrolein  test,  66 
Adamkiewicz  test,  83 
Adenine,  165 
Adrenalin,  184,  199,  200 
Albuminates  (See  Metaproteins) 
Albuminoids,  73,  96 
Albumins,  73,  95 
Alcaptonuria,  157 
Alcohol,  as  protein  precipitant,  89 

utilization  of,  195 
Alkaloidal  reagents,  as  protein  pre- 

cipitants,  89 
Almen  Nylander  test,  42 
Amines  from  amino  acids,  94 
Amino  acid  requirements,   179-181 
Amino  acids,  absorbed,  153 
action  of  formaldehyde  on,  80 
action  of  nitrous  acid  on,  81 
action  with  bases,  80 
addition  of  acids,  80 
as  essential  diet  factors,  112 
as  source  of  urea,  162 
conversion  to  amines,  94 
fate  of,  175 
feeding  with,  181 
from  proteins,   76 
general  properties,  79 
glycogen  formers,  189 
in  blood,  174 


Amino  acids — Cont'd 

isolation  of  optical  isomers,  90 

list  and  formulae,  76-79 

oxidation  of,  81 

per   cent   obtained   from   proteins, 
76 
Amino   sugars,   from   lobster    shells, 

53 
Ammonia  in  urine,  167 
Amylase,  pancreatic,  144 
Anaerobic  respiration,  43 
Arabinose,  34,  50 
Arginine,  source  of  urea,  163 

split  by  alkali,  81 
Arsenic,  21 
Ash,  amount  in  body,  19 

B 

Basterial  action,  intestinal,  149 
Barfoed's  test,  41 
Barley  sugar,  55 
Beri-beri,  197 
Bile,  141,  145-147 

amount  secreted,  145 

cause  of  flow,  145 

composition  of,  146 

effect  on  fat  digestion,  144 

effect  on  tryptic  digestion,  143 

in  fat  absorption,   154 

role  in  digestion,  146 
Bile  pigments,  146 
Bile  salts,  147 
Bilirubin,  146 
Biuret,  from  urea,  161 
Biuret  test,  82 

Birotation    (See   Mutarotation) 
Blood,  121-125   (See  also  Hemoglob- 
in, etc.) 
Blood,  catalytic  activity  of,  102 

coagulation  of,  124 

composition  of,  122 

corpuscles,   composition   of,   122 

enzymes    in,    123 

319 


320 


INDEX 


Blood— Cont'd 

gases,  transference  of,  122 

general  functions  of,  121 

guaiac  test  for,  103 

hydrogen-ion      concentration      of, 
124 

laking  of,  101 

plasma,  composition  of,  122 

platelets,  123 

osmotic  pressure  of,   124 

reaction  of,  123 
Bones,  composition  of,  121 
Brain,  composition  of,  120 

glucosides  found  in,  38 
Breadstuffs,  value  as  foods,   118 
Breakfast  foods,  value  as  foods,  118 
Bromine,  21 
Butter,  68 

Butter,  value  as  food,  116 
Buttermilk,  116 
Butyric   acid,   62 

C 

Calcium,  21 

blood  clotting  dependent  on,  22 

detection  of,  25 

distribution  in  body,  25 

in   clotting    of    milk,    22,    25,    99, 
139 

phosphate  in  bone,  24 
Calorimeter,  192 
Cane  sugar   {See  Saccharose) 
Caprine,  in  brain,  120 
Caramel   formation,    39 
Carbamino  reaction  of  amino  acids, 

80 
Carbohydrates,  19,  27-61 

absorption   of,   153 

amount  in  body,  19 

behavior  with  acids,  39 

behavior  with   alkalies,   38 

can  be   ''built   down,"   37 

classification  of,  34 

combinations -of,  38 

composition  of,  27 

distribution  of,  27 

fats  in  body  {See  Metabolism  of) 

fermentation  of,  44 

individual  groups,  49-61 

interconversion  of,  37 

metabolism  of,   39,  182-189 

metabolism  D:N  ratio,  189 


Carbohydrates — Cont  'd 

optical  activity  of,  28 

origin  of,  35 

osazone  formation,  43 

oxidation  of,  40 

reduction  of,   42 

role  in  body,  182 

role  in  plants  and  animals,  27 

structure  of,  27 

synthesis  in  animals,  36 

synthesis  in  laboratory,  36 

synthesis  in  plants,  35 

tests  for,  40-44 
Carbon,  21,  23 

Carbon    monoxide    hemoglobin,    105 
Cartilage,  composition  of,   121 
Caseinogen,  altered  by  rennin,  138 
Casein,  digested  by  erepsin,  144 

general  properties,  99 
Cataphoresis,   86 
Cellulose,  34,  59 

food  value,  60 

value  to  body  of,  118 
Cerebron,  in  brain,  120 
Cerebrosides,  in  brain,  120 
Cheese,  value  as  food,  117 
Chewing,  importance  of,  127 
Chlorides,   26,   169 
Chlorine,  21 
Chlorophyl,     role     in     carbohydrate 

synthesis,   35 
Cholesterol,  70,  71 

in  bile,  146 

in  brain,   120 
Chromoproteins,  100 
Chyme,  141 

causes  flow  of  bile,  145 
Coagulated    proteins,    73,    110 
Cod  liver  oil,   69 
Collagen,  properties,  96 
Colloids,  84-87 

protective,  87 

Tyndalls  phenomenon,  86 
Conjugated   glucuronates    in    urine, 

159 
Conjugated   proteins,    73,   98-109 
Connective    tissue,    composition   of, 

121 
Cooking,  importance  of,  115 
Copper,  21 
Cream,  116 
Creatine,  in  brain,  120 

in  urine,  167 


INDEX 


321 


Creatinine,  in  urine,  167 
Cretinism,  200 

Cyanhydrin     synthesis     of    carbohy- 
drates,   .^6 
Cystin,  bacterial  destruction  of,  150 

D 

Deficiency  diseases,  198 
Derived  proteins,  109-13  3 
Dextrin,  34 

general  properties  of,  59 

in  salivary  digestion,  129 
Dextrose  {See  Glucose) 
Diabetes  mellitus,   185 
Diet,  choice   of,   118 
Digestion,  general  purpose  of,  126 

in  intestine,  141-150 

in  mouth,  126-130 

in  stomach,  131-140 
Disaecharides,  34,  54-5.7 

Eck's  fistula,  163 

Edestin,  96 

Eggs,  composition  of,  117 

Elastin,   97 

Elements  in  body,  21-26 

Emulsification,  64 

Emulsions,   importance   of,    65 

Energy  exchange,  191 

Energy  requirements,  194 

Enterokinase,  148 

Enzymes,  44-49 

Enzymes  vs.  ' '  Ferments, ' '  46 

Erepsin,  intestinal,  148 

pancreatic,  144 
Ethereal  sulphates  in  urine,  171 
Excretion,  general,  155 
Extractives,  19,  20 
Extractives  in  brain,  120 

F 

Fats,  62-69 

absorption  of,  154 
acetyl  equivalent,  68 
acrolein  test  for,  66 
amounts  in  body,  19,  62 
composition  of,  62 
detection,  66 
distribution,  62 
emulsification  of,  64 
formula  of,  63 
general  properties,  64 


Fats— Cont  'd 

identification    of,    66 

iodine  number,  67 

melting  points  of,  67 

metabolism  of,  189,  190 

rancid,  66 

Reichert-Meissl  number,   67 

saponification  equivalent,   67 

saponification  of,  65 

sources  of,  in  body,  190 

storage  of,  189 

volatile  fatty  acids  in,  67 
Fatty  acids,  62 

formed  from  carbohydrates,  42 
Feces,  composition  and  amount,  150 
Fehling's  test,  chemistry  of,  40 
Fibrinogen,  properties,  96 
Field  of  physiological  chemistrj',  17 
Fluorine,  21,  26 
Food  accessories,  197 
Food,  definition  of,  114 
Foods,  heat  equivalent  of,  193 

specific  dynamic  action  of,  194 
Foodstuffs,  114-119 
Formic  acid,  from  carbohydrates,  38 
Fructose,   28,   34 

conversion  to  other  sugars,  37 

distinction  from  glucose,  52 

distribution,  52 
Fruit  sugar  {See  Fructose) 
Fruits,  value  as  foods,  118 
Furfurol,     formed     from     carbohy- 
drates, 39 

G 

Galactose,  34,  52 

formation  from  glucose,  37 
Gall  stones,  147 
Gastric   digestion,   131-140 
Gastric  juice,  134 

cause  of  flow,  132 
Gastrin,  133 
Gelatine,  properties  of,  97 

in  diet,  180 
Gliadin,   96 
Globulins,   73,  95 
Glucosamine,  53 
Glucose,  34,  51 

conversion  to  other  sugars,  37 

formula  of,  28 

in  blood,   153 

in  normal  urine,  159 

ultimate  fate,  186-187 


322 


INDEX 


Glucosides,  38,  61 

Glucuronates,  conjugated,  formation 

of,  54 
Glucuronic  acid,  properties,  53 
Glutamic  acid,  feeding  with,  182 
Glutelins,  73,  96 
Glycerine,  in  fats,  62 
Glycocoll,  built  in  body,  180 
Glycogen,  34,  182-184 

formed  from  various  sugars,  37 

general  properties,   60 

preparation  of,  60 

sources  of,  186 

stored   in  body,   153,   183    . 
Glycogen,  yields   glucose   on   hydrol- 
ysis, 37 
Glycoproteins,  73,  98 
Glycosuria,  temporary,  186 
Grape  sugar  (See  Glucose) 
Guaiac  test  for  blood,  103 
Guanine,  165 

Guenzburg's  reagent,   135 
Gums,  34,  59 

H 

Haines,  test,  42 

Half  rotation,  33 

Hard  water  for  washing,  66 

Hematin,  106 

Hematoporphyrin,   107 

Hemin  test,  103 

Hemoehromogen,  106 

Hemoglobin,  73,  100-107,   (See  Oxy- 
hemoglobin, etc.) 

Hemoglobin,  absorption  spectrum  of, 
104 
as  Oj  carrier,  100,  122 
CO  derivative  of,  105 
detection  of,  102 
fate  of  in  body,  107 
molecular  weight  of,  101 
source  of  bile  pigments,  147 

Hexoses,  51-54 

Hippuric  acid,  166 

Histones,  73,  97 

Hopkins-Cole  test,    83 

Hormones,  133,  142,  199 

Hydrochloric  acid  of   gastric  juice, 
134,  136 

Hydrogen,  21,  23 

Hydrogen   sulphide   in   sulphur  test, 
24 

Hypoxanthine,  165 


Indol,  94,  150 

Inorganic  materials,  19,  21-26 

metabolism  of,  190-191 
Intestine,  absorption  in,  152 

bacterial  action  in,  149 

digestion  in,  141-150 
Inulin,  34,  59 

Inverting  enzymes,  intestinal,  148 
Iodine,  21,  26 
Iron,   21,  25,   26 
Islands  of  Langerhans,  184 

K 

Keratin,  properties,  96 

Kidneys,  and  blood  sugar,  183,  185 

Lactase,  pancreatic,  145 

Lactose,  34,  37,  56 

Lanolin,  69 

Large  intestine,  secretion  of,  148 

Lavoisier,  191 

Lead,  21 

Lecithoproteins,   73,   109 

Lecithin,  69 

in  brain,  120 
Levulinic  acid,  from  carbohydrates, 

39 
Levulose   (See  Fructose) 
Lipase  of  gastric  juice,  139 
Lithium,  21 
Liver,   and   hemoglobin    destruction, 

25 
Living  material,  characteristics  of, 

18 
Lymph,  64,  125 
Lysine,  need  of,  180 

M 

Magnesium,  21,  25 

Malt  sugar  (See  Maltose) 

Maltase,  in  blood,  153 

pancreatic,  145 
Maltose,  34,  57 
Manganese,  21 
Mannose,  34,  37 
Material  bases,  groups,  19 
Meats  as  foodstuffs,  117 
Mental  activity  and  metabolism,  195 
Metabolism,  173-201 

general  discussion,  173 

in  sleep,  195 

in  starvation,  196-197 

methods  of  study,  173 


INDEX 


323 


Metabolism — Cont  'd 

of  carbohydrates,  182-189 

of  energy,  191-195 

in  disease,  195  ' 

of  fats,  189 

of  inorganic  materials,  190-191 

of  proteins,  174-182 
Metaproteins,  73,  110 

in  gastric  digestion,  136 
Methemoglobin,  105 
Mett's  tubes,  137 
Milk,  104,  115 

souring  of,  116 
Milk  sugar,  {See  Lactose) 
Millon  test,  83 
Minimum,   law   of,    21 
Molisch  reaction,  44 
Monosaccharides,  34,  49-54 
Mouth,  absorption  in,  152 
Mucic  acid  test,  53 
Mucilages,  34,  59 
Mucins,  98,  128 
Mucoids,  98 
Murexid  test,  164 
Muscle,  composition  of,   119 

contraction  of,  119 
Mutarotation,  33 
Myosin,  96 
Myosinogen,   96 

N 

Nerve,  compounds  in,  120 
Neutral  sulphur  in  urine,  170 
Nitrogen,  21,  24 
Nitrogen  balance,  176,  177 
Nuclease,  intestinal,  148 

pancreatic,  145 
Nucleic  acid,  108 

source  of,  164 
Nucleins,  from  nucleoproteins,   108 
Nucleoproteins,  73,  107,  109 
Nuts,  value  as  food,  118 

O 

Oils,  64 

Oleic  acid,  62 

Oleomargarine,  69 

Optical  activity,  32,  33 

Optical  isomers,  number  possible,  34 

Optimum  temperature  for  enzymes, 

47 
Osazones,  43 
Oxalic  acid,  from  carbohydrates,  38 


Oximes,  37 
Oxygen,  21,  23 

Oxyhemoglobin,  spectrum,  104 
crystallization  of,  102 

P 
Palmitic  acid,  62 

Pancreas  and  sugar  metabolism,  184 
Pancreatic  juice,  141-142 
Paracasein,  produced  by  rennin,  138 
Pentoses,  39,  49 
Pepsin,  137,  138,  139,  141 
Peptids,  73,  112 

behavior  with  enzjones,  93 

precipitation  of,  93 

properties  of,  92 

synthesis  of,  90-92 
Peptones,  73,  111 

in  blood,  175 

in  peptic   digestion,   138 
Phenol,  gives  Millon 's  test,  83 
Phloridzin  diabetes,  185 
Phosphates,  24 

in  urine,  170 
Phosphatides,  19,  69-71 
Phospholipins,  in  brain,  120 
Phosphoproteins,  73,  98,  99 
Phosphorus,   21,   24 
Pituitary,  and  metabolism,  200 
Polariscope,  31 
Polarized  light,  29 
Polysaccharides,  34,  57-61 
Potassium,  21,  24,  25 
Prolamines,  73,  96 
Proline,  built  in  body,  180 

in  prolamines,  96 
Protamines,  73,  97 
Proteans,  73,  109 
Proteins,  19,  72-113 

absorption  of,  153 

amount  in  body,  19 

amount  required,  176-181 

classification  of,  73 

color  tests  for,  82-84 

elementary  composition,  72 

evidence  for  structure  of,  92 

form  in  which  absorbed,  174 

general   reactions,   82-89 

hydrolysis  of,   75 

hydrolysis   not   symmetrical,   93 

importance,   72 

individual   groups,   95-113 

metabolism  of,  174-182 


324 


INDEX 


Proteins — Cont  'd 

molecular  weight  of,  75 

peptid  linkage  in,  90 

precipitation  reactions,  84-89 

preparation  of,  74 

putrefaction  of,  93 

specific  nature  of,  182 

storage  of,   175,   176 

structure  of,  89-93 
Proteoses,  73,  111 

in  blood,  174 

in  peptic  digestion,  138 
Protoplasm,   properties   of,   18 
Ptomaines,  94,  149 
Ptyalin,  128 
Puncture  diabetes,  183 
Purine  bases,  from  nucleic  acid,  108 

sources  of  uric  acid,  164 

synthesis  in  body,  166 
Putrefaction,  intestinal,  149 

intestinal,  reduced  by  bile,  146 

of  proteins,  93 
Pylorus,  control  of,  140 
Pyrimidene  bases,  from  nucleic  acid, 

108 
Pyruvic   acid,   81,   176 

E 
Rennin,  138,  139 

pancreatic,  144 
Respiratory  quotient,  186 
Rhamnose,  27 
Ribose,  34 

S 

Saccharose,  34,  54-56 
Saliva,  127-130 
Saponification  of  fats,  65 
Secondary   protein   derivatives,   111- 

113 
Secretin,  142 

Sexual  glands,  and  metabolism,  200 
Silicon,  21 
Silver,  21       • 

Simple  proteins,  73,  95-98 
Skatol,  94,  149 
Skin,  125 
Soap,  as  cleansing  agent,  66 

as  emulsifier  of  fats,  64 
Soda  lime,  test  for  N,  24 
Sodium,  21,  24,  25 
Sodium  chloride  in  diet,  25 

phosphate,  24 


Specific    dynamic    action    of    foods, 

194 
Specific  rotation,  29 
Spleen,  contains  iron,  25 
Starch,  34,  35,  58-59 

digested  by  pancreatic  juice,  144 

digested  by  saliva,  129 
Starvation,  196-197 
Steapsin,  pancreatic,  144 
Stearic  acid,  62 
Stokes  reagent,  101 
St.  Martin,  Alexis,  132 
Stomach,  absorption  in.  152 

digestion  in,   131-140 

importance   of,   131 

why  not  digested.  140 
Succus  entericus,  141,  147 
Sucrose,  (See  Saccharose) 
Sugar  in  blood,  182 
Sugar  center  in  brain.  183 
Sulphates,  detection  of,  24 

in  urine,  170 
Sulphur,   21.   24 

in  proteins,  test  for.  84 
Suprarenals,  and  blood  sugar,  184 

T 

Teeth,  composition  of,  121 
Temperature    of    body,    control    of. 

199 
Temporary  glycosuria,  186 
Thymus,  and  metabolism,  201 
Thyroid,  and  metabolism,  200 

contains  iodine,  26 
Triolein,  distribution,  68 
Tripalmitin,  distribution,  68 
Tri stearin,  properties,  68 
Trypsin,   143 
Trypsinogen,  143 
Tryptophane,  body's  need  of,  180 

destruction  of,  by  bacteria,  149 

in  Adamkiewicz  test.  84 
Tyndall's  phenomenon,  86 
Tyrosine,    bacterial    destruction    of, 
149 

in  Millon's  test,  83 

need  of,  180 

U 

Unoxidized  sulphur,  test  for,  24 
Urea,  161-163 

physiological  action  of,  163 

sources  of,  162 


INDEX 


325 


Urease,  162 

Uric  acid,  163-166 

endogenous,  165 

exogenous,  165 

sources  of,  164 

variations  in  disease,  166 
Uriease,  missing  in  man,  165 
Urine,   155-172 

ammonia  in,  167 

carbonates  in,  171 

chlorides  in,  169 

color  and  transparency  of,  157 

consistency  of,  158 

creatine  and  creatinine  in,  167-169 

fermentation  of,  159 

hippuric  acid  in,  166 

odor  of,  158 

optical  activity  of,  159 

pathological  constituents  of,  172 
{See  Index  of  Laboratory 
Work) 

phosphates  in,  170 

reaction  of,  160 

reducing  power,  159 

sediments,  157 

specific  gravity  of,  158 

sulphates  in,  170 

taste  of,  158 


Urine — Cont'd 

total  solids  in,  158 

toxic  properties  of,  160 

urea  in,  161-163 

uric  acid  in,   163-166 

volume   of,   156 
Urobilin,    source,    146 
Urochrome,  157 

V 

Value  of  physiological  chemistry,  18 
Vegetables,  as  foodstuffs,  117 
Vitamines,   197-199 
Vitellin,  100 
Vividiffusion,  175 

W 

Water,  19,  26 

absorbed  in  large  intestine,  154 
Waxes,  63 
Work,  and  nitrogen  excretion,  179 

X 

Xanthine,   165 
Xanthoproteic  test,  83 
Xylose,  34,  50 

Z 
Zein  in  diet,  180 


INDEX  OF  LABORATORY  WORK 


Absorption  spectra,  244-247 

Acetone  in  urine,  306 

Acrolein  test,  229 

Adamkiewicz  test,  233 

Albumin    crystals,    preparation    of, 

237 
Albuminoids,  240,  241 
Albumins,  test  with,  237 
Amino  acids,  257 
Atomic  weights,  table  of,  313 


Barfoed's  test,  218 
Benzidene  test,  248 
Bile,  270-272 
Biliary  calculi,  271 
Biuret  test,  232 
Blood,  243-250 

benzidene  test  for,  248 

catalase  in,  249 

chemical  tests  for,  247-249 

serum,  inorganic  tests,  212 
Bone,  inorganic  tests,  213 


Calcium,  test  for  214 

Cane  sugar,  (See  Saccharose) 

Caramel  test,  219 

Carbohydrates,  277-6 

Carbon,  test  for,  210 

Carbonates,  test  for,  214 

Caseinogen,  250 

Catalase  in  blood,  249 

Chlorides,  test  for,  213 

Cholesterin  in  gall  stones,  272 

Coagulated  proteins,  254-255 

Coagulation  temperature  of  proteins, 

238 
CO  hemoglobin,  246 
Collagen,  241 
Congo  red  test,  265 
Conjugated  proteins,  242-253 
Cystin,   259 


D 

Derived  proteins,  253-257 
Dextrines,  225 
Dextrose,  217-221 
Digestion,  in  intestine,  269-272 

in  mouth,   260-263 

in  stomach,  264-268 
Disaccharides,  222-224 

E 
Elastin,  241 
Elements,  210-216 
Emulsification,  228 

F 

Fats,  227-230 

intestinal  digestion  of,  270 
Fehling's  test,  qualitative,  217 

quantitative,  304 
Fermentation  test,  220 
Fibrin,  239 

G 

Galactose,  22 

Gall  stones,  271 

Gastric  juice,  composition  of,  264 

digestive  action,  266-268 
Gelatine,  241 

General  instructions,  208-209 
Gliadin,  240 
Globin,  249 

Globulins,  tests  with,  238 
Glutelins,  240 
Glutenin,  240 

Glycogen,  preparation  and  tests,  226 
Glycoproteins,  242-243 
Gmelin  test,  271 
Guaiae  test,  248 
Guenzburg's  test,  265 

H 

Haines'  test,  218 
Heller's  test,   300 
Hematin,  246 
Hematoporphyrin,  247 


326 


INDEX 


327 


Hemin  crystals,  247 
Hemochromogen,  247 
Hemoglobin,  crystals  of,  247 
Hemoglobins,  243-250 

absorption,  spectra  of,  244-247 
Histones,  241 
Hopkins-Cole  test,  233 
Hydrochloric   acid   of   gastric  juice, 

264-266 
Hydrogen,  test  for,  210 


Inorganic  materials,  210-216 

tests  for,  213-216 
Intestinal  juice,  270 
Inversion  of  saccharose,  223 
Iodine  test,  225 
Iodoform  test  for  acetone,  307 
Iron,  test  for,  215 


Jaffe's  test,  277 


K 


Keratin,  240 


Lactose,  tests  with,  224 
Lecithin,  230 
Lecithoproteins,  253 
Levulose,  tests  with,  221 
Leucine,  258 

M 

Magnesium,  test  for,  214 
Maltose,  tests  with,  223 
Materials,  202-207 
Metaproteins,  253-254 
Methemoglobin,  246 
Mett's  tubes,  266 
Milk,  250 

coagulation  of  by  rennin,  267 
Millon's  test,  233 
Molisch  test,  219 

on  proteins,  234 
Motor  power  of  stomach,  268 
Mucic  acid  test,  221 
Mucin,  242 
Murexid  test,  276 

Muscle  extract,  preparation  of,  211 
Muscle  residue,  preparation  of,  212 
Myosin,  239 


N 

Neutral  oil,  preparation  of,  227 
Nitrogen,  test  for,  211 
Nucleoproteins,  251-253 
Nylander's  test,  219 

O 

Optical  activity,  219 
Orcin  test,  222 
Oxygen,  210 

P 

Pancreatic   digestion,   269-272 

Pentoses,  222 

Peptids,  257 

Peptones,  256 

Pettenkofer's  test,  271 

Phenylhydrazine   test,    219 

Phloroglucin  test,   222 

Phosphates,  test  for,  214 

Phosphatides,  230,  231 

Phosphoproteins,  250,  251 

Pipettes  and  burettes,  use  of,  280 

Polysaccharides,  224-226 

Potassium,  test  for,  216 

Precipitation  tests,  for  proteins,  234 

Prolamines,   240 

Protamines,  242 

Proteans,  253 

Proteins,  252-259 

Adamkiewiez  test  for,  233 
biuret  test,  232 
color  tests  for,  232-234 
gastric  digestion  of,  266 
intestinal  digestion  of,  269 
Millon's  test  for,  233 
precipitation  tests  for,  234 
xanthoproteic  test  for,  233 

Proteoses,  255 

Ptyalin,  action  of,  261 

B 

Rothera's  test,  307 

S 

Saccharose,  222,  223 
Saliva,  260,  261 
Salivary  digestion,  260-263 
Saponification,  229 
Sodium,  test  for,  215 
Solubility,    to    test    solubility    of    a 
substance,  217 


328 


INDEX 


Special  reagents,  308-312 

Specific  rotation,  220 

Standard  acid  and  alkali,  282-286 

Starch,  224,  225 

Starch,  digestion  by  ptyalin,  261-263 

Stomach,   absorption   from,   268 

digestion  in,  264-268 
Succus  entericus,  270 
Sucrose,  (See  Saccharose) 
Sulphates,  test  for,  214 
Sulphur,   test   for,   211 

T 

Toepfer's  test,  265 
Tyrosine,  258 

U 

Uffelman's  test,  265 
Urine,  273-307 

acetone  bodies  in,  306-307 

acidity,  estimation  of  (Folin),  286 

albumin  in,  300-301 

ammonia,  qualitative  test,  244 
quantitative  (clinical),  292 
quantitative   (Folin),  290 

carbohydrates  in,  303-306 

carbonates,  qualitative,  274 

chlorides,  qualitative,  273 
quantitative  (Volhard),  297 

collection,  278 

creatinine,    quantitative     (Folin), 
295 

creatinine,  reactions  of,  277 

globulins  in,  301 

hippuric  acid,  294 

indican,   278,   296 

metaprotein,  302 

nucleoprotein,  302 

optical  activity  of,  306 


Urine — Cont  'd 

oxalic  acid,  277,  297 
pathological,  299-307 
phosphates,  qualitative,  273 

quantitative,  299 
pigments,   278 
preservation  of,  278 
proteins,  quantitative,  301 

tests  for,  299-303 
proteoses  and  peptones,  303 
purine  bases,  295 

reactions  of,  276,  277 
qualitative  tests,  273-278 
specific  gravity,  279 
sugar   in,    fermentation    test,    306 

quantitative    (Benedict),    305 

quantitative   (Fehling),  304 
sulphates,  qualitative,  274 

quantitative,  298 
total  nitrogen  (Kjeldahl),  287 
total  solids,  281 
urea,  preparation  of,  275 

quantitative  (urease),  292 
uric  acid,  preparation  of,  275.  276 

quantitative,    (Folin-SchafPer), 
293 
volume,  279 


U 


Uroehrome,  278 
Uroerythrin,  278 

Vitellin,   251 

Weyl's  test,  277 

X 
Xanthoproteic  test,   233 


W 


